Fvii or fviia gla domain variants

ABSTRACT

Gla domain variants of human Factor VII or human Factor VIIa, comprising 1-15 amino acid modifications relative to human Factor VII or human Factor VIIa, wherein a hydrophobic amino acid residue has been introduced by substitution in position 34; or having an amino acid substitution in position 36; and use of the variants for the treatment of intracerebral haemorrhage (ICH) or trauma.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of International PatentApplication No. PCT/DK2004/000428 filed on Jun. 18, 2004, which claimsthe benefit of U.S. Provisional Patent Application Ser. No. 60/479,780filed on Jun. 19, 2003 and of Denmark Patent Application No. PA 200400930 filed on Jun. 15, 2004, the disclosures of each of which areincorporated by reference herein in their entirety for all purposes.

FIELD OF THE INVENTION

The present invention relates to novel Gla domain variants of FactorFVII (FVII) or Factor VIIa (FVIIa) polypeptides, as well as the use ofsuch polypeptide variants in therapy, in particular for the treatment ofa variety of coagulation-related disorders.

BACKGROUND OF THE INVENTION

Blood coagulation is a process consisting of a complex interaction ofvarious blood components (or factors) that eventually results in afibrin clot. Generally, the blood components participating in what hasbeen referred to as the “coagulation cascade” are proenzymes orzymogens, i.e. enzymatically inactive proteins that are converted intoan active form by the action of an activator. One of these coagulationfactors is FVII.

FVII is a vitamin K-dependent plasma protein synthesized in the liverand secreted into the blood as a single-chain glycoprotein with amolecular weight of 53 kDa (Broze & Majerus, J. Biol. Chem. 1980;255:1242-1247). The FVII zymogen is converted into an activated form(FVIIa) by proteolytic cleavage at a single site, R152-I153, resultingin two chains linked by a single disulfide bridge. FVIIa in complex withtissue factor (FVIIa complex) is able to convert both factor IX (FIX)and factor X (FX) into their activated forms, followed by reactionsleading to rapid thrombin production and fibrin formation (Østerud &Rapaport, Proc Natl Acad Sci USA 1977; 74:5260-5264).

FVII undergoes post-translational modifications, including vitaminK-dependent carboxylation resulting in ten γ-carboxyglutamic acidresidues in the N-terminal region of the molecule. Thus, residues number6, 7, 14, 16, 19, 20, 25, 26, 29 and 35 shown in SEQ ID NO:1 areγ-carboxyglutamic acid residues in the Gla domain important for FVIIactivity. Other post-translational modifications include sugar moietyattachment at two naturally occurring N-glycosylation sites at position145 and 322, respectively, and at two naturally occurringO-glycosylation sites at position 52 and 60, respectively.

The gene coding for human FVII (hFVII) has been mapped to chromosome 13at q34-qter 9 (de Grouchy et al., Hum Genet 1984; 66:230-233). Itcontains nine exons and spans 12.8 Kb (O'Hara et al., Proc Natl Acad SciUSA 1987; 84:5158-5162). The gene organisation and protein structure ofFVII are similar to those of other vitamin K-dependent procoagulantproteins, with exons 1a and 1b encoding for signal sequence; exon 2 thepropeptide and Gla domain; exon 3 a short hydrophobic region; exons 4and 5 the epidermal growth factor-like domains; and exon 6 through 8 theserine protease catalytic domain (Yoshitake et al., Biochemistry 1985;24: 3736-3750).

Reports exist on experimental three-dimensional structures of hFVIIa(Pike et al., Proc Natl Acad Sci USA, 1999; 96:8925-30 and Kemball-Cooket al., J. Struct. Biol., 1999; 127:213-223); of hFVIIa in complex withsoluble tissue factor using X-ray crystallographic methods (Banner etal., Nature, 1996; 380:41 and Zhang et al., J. Mol. Biol., 1999; 285:2089); and of smaller fragments of hFVII (Muranyi et al., Biochemistry,1998; 37:10605 and Kao et al., Biochemistry, 1999; 38:7097).

Relatively few protein-engineered variants of FVII have been reported(Dickinson & Ruf, J Biol Chem, 1997;272:19875-19879; Kemball-Cook etal., J Biol Chem, 1998; 273:8516-8521; Bharadwaj et al., J Biol Chem,1996; 271:30685-30691; Ruf et al., Biochemistry, 1999; 38:1957-1966).

Reports exist on expression of FVII in BHK or other mammalian cells (WO92/15686, WO 91/11514 and WO 88/10295) and co-expression of FVII andkex2 endoprotease in eukaryotic cells (WO 00/28065).

Commercial preparations of recombinant human FVIIa (rhFVIIa) are soldunder the trademark NovoSeven®. NovoSeven® is indicated for thetreatment of bleeding episodes in hemophilia A or B patients. NovoSeven®is the only rhFVIIa for effective and reliable treatment of bleedingepisodes currently available on the market.

Mayer (Stroke, 2003, 34:224-229) speculated that ultra-early hemostatictreatment of intracerebral haemorrhage (ICH), given within 3-4 hours ofonset, may arrest bleeding and minimize hematoma growth after ICH. OnJun. 22, 2004, it was reported in a stock exchange announcement by NovoNordisk (Denmark) that NovoSeven® was found to provide a significantlyimproved neurological and functional outcome in the treatment of ICH.However, it was also reported that the treatment was associated with anon-significant increase in thromboembolic events.

An inactive form of FVII in which arginine 152 and/or isoleucine 153 aremodified has been reported in WO 91/11514. These amino acids are locatedat the activation site. WO 96/12800 describes inactivation of FVIIa by aserine proteinase inhibitor. Inactivation by carbamylation of FVIIa atthe (α-amino acid group I153 has been described by Petersen et al., EurJ Biochem, 1999;261 :124-129. The inactivated form is capable ofcompeting with wild-type FVII or FVIIa for binding to tissue factor andinhibiting clotting activity. The inactivated form of FVIIa is suggestedto be used for treatment of patients suffering from hypercoagulablestates, such as patients with sepsis or at risk of myocardial infarctionor thrombotic stroke.

In connection with treatment of uncontrolled bleedings such as trauma itis believed that FVIIa is capable of activating FX to FXa withoutbinding to tissue factor, and this activation reaction is believed tooccur primarily on activated blood platelets (Hedner et al. BloodCoagulation & Fibrinolysis, 2000; 11; 107-111). However, hFVIIa orrhFVIIa has a low activity towards FX in the absence of tissue factorand, consequently, treatment of uncontrolled bleeding, for example intrauma patients, requires relatively high and multiple doses of hFVIIaor rhFVIIa. Therefore, in order to treat uncontrolled bleedings moreefficiently (to minimize blood loss) there is need for improved FVIIamolecules which possess a high activity toward FX in the absence oftissue factor. Such improved FVIIa molecules should exhibit a loweredclotting time (faster action/increased clotting activity) as compared torhFVIIa when administered in connection with uncontrolled bleedings.

Gla domain variants of FVII/FVIIa have been disclosed in WO 99/20767,U.S. Pat. No. 6,017,882 and WO 00/66753, where some residues located inthe Gla domain were identified as being important for phospholipidmembrane binding and hence FX activation. In particular, it was foundthat the residues 10 and 32 were critical and that increasedphospholipid membrane binding affinity, and hence increased FXactivation, could be achieved by performing the mutations P10Q and K32E.In particular, it was found that FX activation was enhanced as comparedto rhFVIIa at marginal coagulation conditions, such as under conditionswhere a low level of tissue factor is present.

WO 01/58935 discloses a new strategy for developing FVII or FVIIamolecules having inter alia an increased half-life by means of directedglycosylation or PEGylation.

WO 03/093465 discloses FVII or FVIIa variants having certainmodifications in the Gla domain and having one or more N-glycosylationsites introduced outside the Gla domain.

WO 2004/029091 discloses FVII or FVIIa variants having certainmodifications in the tissue factor binding site.

The present inventors have now identified further residues in the Gladomain which further increase the phospholipid membrane binding affinityand hence further increase FX activation. The FVII or FVIIa variants ofthe invention may also exhibit reduced tissue factor binding affinity.

The object of the present invention is to provide improved FVII or FVIIamolecules (FVII or FVIIa variants) which are capable of activating FX toFXa more efficiently than hFVIIa, rhFVIIa or [P10Q+K32E]rhFVIIa. Inparticular, it is an object of the present invention to provide improvedFVII or FVIIa molecules (FVII or FVIIa variants) which are capable ofactivating FX to FXa more efficiently than hFVIIa, rhFVIIa or[P10Q+K32E]rhFVIIa in the absence of tissue factor. These objects areaddressed by the FVII or FVIIa variants provided herein.

BRIEF DISCLOSURE OF THE INVENTION

In a first aspect the present invention relates to a Factor VII (FVII)or Factor VIIa (FVIIa) polypeptide variant having an amino acid sequencecomprising 1-15 amino acid modifications relative to human Factor VII(hFVII) or human Factor VIIa (hFVIIa) with the amino acid sequence shownin SEQ ID NO:1, wherein a hydrophobic amino acid residue has beenintroduced by substitution in position 34.

In a second aspect the invention relates to a Factor VII (FVII) orFactor VIIa (FVIIa) polypeptide variant having an amino acid sequencecomprising 1-15 amino acid modifications relative to human Factor VII(hFVII) or human Factor VIIa (hFVIIa) with the amino acid sequence shownin SEQ ID NO:1, wherein the amino acid sequence comprises an amino acidsubstitution in position 36.

In a third aspect the invention relates to a Factor VII (FVII) or FactorVIIa (FVIIa) polypeptide variant having an amino acid sequencecomprising 3-15 amino acid modifications relative to human Factor VII(hFVII) or human Factor VIIa (hFVIIa) having the amino acid sequenceshown in SEQ ID NO:1, wherein amino acid sequence comprises an aminoacid substitution in positions 10 and 32 and at least one further aminoacid substitution in a position selected from the group consisting ofpositions 74, 77 and 116.

Further aspects of the invention relate to a nucleotide sequenceencoding the polypeptide variants of the invention, an expression vectorcomprising the nucleotide sequence, and a host cell comprising thenucleotide sequence or expression vector.

Still further aspects of the invention relate to a pharmaceuticalcomposition comprising the polypeptide variants of the invention, use ofthe polypeptide variants of the invention or the pharmaceuticalcomposition of the invention as a medicament, as well as methods oftreatment using the polypeptide variants or pharmaceutical compositionsof the invention. In a particular aspect, the polypeptide variants ofthe invention are used for the treatment of intracerebral haemorrhage ortraumatic brain injury.

Further aspects of the present invention will be apparent from thedescription below as well as from the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the clotting time vs. concentration for variants of theinvention when assayed in the “Whole Blood Assay”.

FIG. 2 shows the maximum tissue factor-dependent thrombin generationrate for variants of the invention determined in the “ThrombogramAssay”.

FIG. 3 shows the maximum phospholipid-dependent thrombin generation ratefor variants of the invention determined in the “Thrombogram Assay”.

FIG. 4 is a thrombogram showing the phospholipid-dependent clottingactivity of a variant of the invention (P10Q+K32E+A34E+R36E+T106N+V253N)compared to rhFVIIa (NovoSeven®).

FIG. 5 is a thrombogram showing the tissue factor-dependent clottingactivity of a variant of the invention (P10Q+K32E+A34E+R36E+T106N+V253N)compared to rhFVIIa (NovoSeven®).

DETAILED DISCLOSURE OF THE INVENTION

Definitions

In the context of the present description and claims the followingdefinitions apply:

The term “FVII” or “FVII polypeptide” refers to a FVII molecule providedin single chain form. One example of a FVII polypeptide is the wild-typehuman FVII (hFVII) having the amino acid sequence shown in SEQ ID NO:1.It should be understood, however, that the term “FVII polypeptide” alsocovers hFVII-like molecules, such as fragments or variants of SEQ IDNO:1, in particular variants where the sequence comprises at least one,such as up to 15, preferably up to 10, amino acid modifications ascompared to SEQ ID NO:1.

The term “FVIIa” or “FVIIa polypeptide” refers to a FVIIa moleculeprovided in its activated two-chain form. When the amino acid sequenceof SEQ ID NO:1 is used to describe the amino acid sequence of FVIIa itwill be understood that the peptide bond between R152 and I153 of thesingle-chain form has been cleaved, and that one of the chains comprisesamino acid residues 1-152, the other chain comprises amino acid residues153-406.

The terms “rFVII” and “rFVIIa” refer to FVII and FVIIa polypeptidesproduced by recombinant techniques.

The terms “hFVII” and “hFVIIa” refer to human wild-type FVII and FVIIa,respectively, having the amino acid sequence shown in SEQ ID NO:1

The terms “rhFVII” and “rhFVIIa” refer to human wild-type FVII andFVIIa, having the amino acid sequence shown in SEQ ID NO:1, produced byrecombinant means. An example of rhFVIIa is NovoSeven®.

When used herein, the term “Gla domain” is intended to cover amino acidresidues 1 to 45 of SEQ ID NO:1.

Accordingly, the term “position located outside the Gla domain” coversamino acid residues 46-406 of SEQ ID NO:1.

The abbreviations “FX”, “TF” and “TFPI” mean Factor X, Tissue Factor andTissue Factor Pathway Inhibitor, respectively.

The term “protease domain” is used about residues 153-406 counted fromthe N-terminus.

The term “catalytic site” is used to mean the catalytic triad consistingof S344, D242 and H193 of the polypeptide variant.

The term “parent” is intended to indicate the molecule to bemodified/improved in accordance with the present invention. Although theparent polypeptide to be modified by the present invention may be anyFVII or FVIIa polypeptide, and thus be derived from any origin, e.g. anon-human mammalian origin, it is preferred that the parent polypeptideis hFVII or hFVIIa.

A “variant” is a polypeptide which differs in one or more amino acidresidues from its parent polypeptide, normally in 1-15 amino acidresidues (e.g. in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15amino acid residues), such as in 1-10 amino acid residues, e.g. in 1-8,1-6, 1-5 or 1-3 amino acid residues. Normally, the parent polypeptide ishFVII or hFVIIa.

The term “conjugate” (or interchangeably “conjugated polypeptide”) isintended to indicate a heterogeneous (in the sense of composite orchimeric) molecule formed by the covalent attachment of one or morepolypeptides to one or more non-polypeptide moieties such as polymermolecules, lipophilic compounds, sugar moieties or organic derivatizingagents. Preferably, the conjugate is soluble at relevant concentrationsand conditions, i.e. soluble in physiological fluids such as blood.Examples of conjugated polypeptides of the invention includeglycosylated and/or PEGylated polypeptides.

The term “covalent attachment” or “covalently attached” means that thepolypeptide variant and the non-polypeptide moiety are either directlycovalently joined to one another, or else are indirectly covalentlyjoined to one another through at least one intervening moiety such as abridge, spacer, or linkage moiety.

The term “non-polypeptide moiety” is intended to mean a molecule,different from a peptide polymer composed of amino acid monomers andlinked together by peptide bonds, which molecule is capable ofconjugating to an attachment group of the polypeptide variant of theinvention. Preferred examples of such molecules include polymermolecules, sugar moieties, lipophilic compounds or organic derivatizingagents. When used in the context of a conjugated variant of theinvention it will be understood that the non-polypeptide moiety islinked to the polypeptide part of the conjugated variant through anattachment group of the polypeptide. As explained above, thenon-polypeptide moiety can be directly or indirectly covalently joinedto the attachment group.

A “polymer molecule” is a molecule formed by covalent linkage of two ormore monomers, wherein none of the monomers is an amino acid residue,except where the polymer is human albumin or another abundant plasmaprotein. The term “polymer” may be used interchangeably with the term“polymer molecule”. The term is also intended to cover carbohydratemolecules attached by in vitro glycosylation, i.e. a syntheticglycosylation performed in vitro normally involving covalently linking acarbohydrate molecule to an attachment group of the polypeptide variant,optionally using a cross-linking agent.

The term “sugar moiety” is intended to indicate acarbohydrate-containing molecule comprising one or more monosaccharideresidues, capable of being attached to the polypeptide variant (toproduce a polypeptide variant conjugate in the form of a glycosylatedpolypeptide variant) by way of in vivo glycosylation. The term “in vivoglycosylation” is intended to mean any attachment of a sugar moietyoccurring in vivo, i.e. during posttranslational processing in aglycosylating cell used for expression of the polypeptide variant, e.g.by way of N-linked and O-linked glycosylation. The exact oligosaccharidestructure depends, to a large extent, on the glycosylating organism inquestion.

An “N-glycosylation site” has the sequence N—X—S/T/C, wherein X is anyamino acid residue except proline, N is asparagine and S/T/C is eitherserine, threonine or cysteine, preferably serine or threonine, and mostpreferably threonine. Preferably, the amino acid residue in position +3relative to the asparagine residue is not a proline residue.

An “O-glycosylation site” is the OH-group of a serine or threonineresidue.

The term “attachment group” is intended to indicate a functional groupof the polypeptide variant, in particular of an amino acid residuethereof or a carbohydrate moiety, capable of attaching a non-polypeptidemoiety such as a polymer molecule, a lipophilic molecule, a sugar moietyor an organic derivatizing agent. Useful attachment groups and theirmatching non-polypeptide moieties are apparent from the table below.Conjugation Attachment Examples of non- method/- group Amino acidpolypeptide moiety Activated PEG Reference —NH₂ N-terminal, Polymer,e.g. PEG, mPEG-SPA Nektar Therapeutics Lys with amide or imineTresylated mPEG Delgado et al, Critical group reviews in TherapeuticDrug Carrier Systems 9(3,4): 249-304 (1992) —COOH C-terminal, Polymer,e.g. PEG, mPEG-Hz Nektar Therapeutics Asp, Glu with ester or amide groupCarbohydrate In vitro coupling moiety —SH Cys Polymer, e.g. PEG,PEG-vinylsulphone Nektar Therapeutics with disulfide, PEG-maleimideDelgado et al, Critical maleimide or vinyl reviews in sulfone groupTherapeutic Drug Carbohydrate In vitro coupling Carrier Systems moiety9(3,4): 249-304 (1992) —OH Ser, Thr, Sugar moiety In vivo O-linked Lys,OH— glycosylation PEG with ester, ether, carbamate, carbonate —CONH₂ Asnas part Sugar moiety In vivo N- of an N- glycosylation glycosylationPolymer, e.g. PEG site Aromatic Phe, Tyr, Carbohydrate In vitro couplingresidue Trp moiety —CONH₂ Gln Carbohydrate In vitro coupling Yan andWold, moiety Biochemistry, 1984, Jul 31; 23(16): 3759-65 AldehydeOxidized Polymer, e.g. PEG, PEGylation Andresz et al., 1978, Ketoneoligo- PEG-hydrazide Macromol. Chem. saccharide 179: 301, WO 92/16555,WO 00/23114 Guanidino Arg Carbohydrate In vitro coupling Lundblad andNoyes, moiety Chemical Reagents for Protein Modification, CRC PressInc., Florida, USA Imidazole His Carbohydrate In vitro coupling As forguanidine ring moiety

For in vivo N-glycosylation, the term “attachment group” is used in anunconventional way to indicate the amino acid residues constituting aN-glycosylation site (with the sequence N—X—S/T/C as indicated above).Although the asparagine residue of the N-glycosylation site is the oneto which the sugar moiety is attached during glycosylation, suchattachment cannot be achieved unless the other amino acid residues ofthe N-glycosylation site are present.

Accordingly, when the non-polypeptide moiety is a sugar moiety and theconjugation is to be achieved by in vivo N-glycosylation, the term“amino acid residue comprising an attachment group for a non-polypeptidemoiety” as used in connection with alterations of the amino acidsequence of the polypeptide is to be understood as meaning that one ormore amino acid residues constituting an in vivo N-glycosylation siteare to be altered in such a manner that a functional in vivoN-glycosylation site is introduced into the amino acid sequence.

In the present application, amino acid names and atom names (e.g. CA,CB, CD, CG, SG, NZ, N, O, C, etc) are used as defined by the ProteinDataBank (PDB) (www.pdb.org) based on the IUPAC nomenclature (IUPACNomenclature and Symbolism for Amino Acids and Peptides (residue names,atom names, etc.), Eur. J. Biochem., 138, 9-37 (1984) together withtheir corrections in Eur. J. Biochem., 152, 1 (1985)).

The term “amino acid residue” is intended to include any natural orsynthetic amino acid residue, and is primarily intended to indicate anamino acid residue contained in the group consisting of the 20 naturallyoccurring amino acids, i.e. selected from the group consisting ofalanine (Ala or A), cysteine (Cys or C), aspartic acid (Asp or D),glutamic acid (Glu or E), phenylalanine (Phe or F), glycine (Gly or G),histidine (His or H), isoleucine (Ile or I), lysine (Lys or K), leucine(Leu or L), methionine (Met or M), asparagine (Asn or N), proline (Proor P), glutamine (Gln or Q), arginine (Arg or R), serine (Ser or S),threonine (Thr or T), valine (Val or V), tryptophan (Trp or W), andtyrosine (Tyr or Y) residues.

The terminology used for identifying amino acid positions is illustratedas follows: G124 indicates that position 124 is occupied by a glycineresidue in the amino acid sequence shown in SEQ ID NO: 1. G124Rindicates that the glycine residue of position 124 has been substitutedwith an arginine residue. Alternative substitutions are indicated with a“/”, e.g. N145S/T means an amino acid sequence in which asparagine inposition 145 is substituted with either serine or threonine. Multiplesubstitutions are indicated with a “+”, e.g. K143N+N145S/T means anamino acid sequence which comprises a substitution of the lysine residuein position 143 with an asparagine residue and a substitution of theasparagine residue in position 145 with a serine or a threonine residue.Insertion of an additional amino acid residue, e.g. insertion of analanine residue after G124, is indicated by G124GA. Insertion of twoadditional alanine residues after G124 is indicated by G124GAA, etc.When used herein, the term “inserted in position X” or “inserted atposition X” means that the amino acid residue(s) is (are) insertedbetween amino acid residue X and X+1. A deletion of an amino acidresidue is indicated by an asterix. For example, deletion of the glycineresidue in position 124 is indicated by G124*.

Unless otherwise indicated, the numbering of amino acid residues madeherein is made relative to the amino acid sequence of the hFVII/hFVIIapolypeptide (SEQ ID NO:1).

The term “differs from” as used in connection with specific mutations isintended to allow for additional differences being present apart fromthe specified amino acid difference. For instance, in addition to themodifications performed in the Gla domain aiming at increasing the FXactivation, the polypeptide may contain other modifications that are notnecessarily related to this effect.

Thus, in addition to the amino acid modifications disclosed herein, itwill be understood that the amino acid sequence of the polypeptidevariant of the invention may, if desired, contain other alterations,i.e. other substitutions, insertions or deletions. These may, forexample, include truncation of the N— and/or C-terminus by one or moreamino acid residues (e.g. by 1-10 amino acid residues), or addition ofone or more extra residues at the N— and/or C-terminus, e.g. addition ofa methionine residue at the N-terminus or introduction of a cysteineresidue near or at the C-terminus, as well as “conservative amino acidsubstitutions”, i.e. substitutions performed within groups of aminoacids with similar characteristics, e.g. small amino acids, acidic aminoacids, polar amino acids, basic amino acids, hydrophobic amino acids andaromatic amino acids.

Examples of such conservative substitutions are shown in the belowtable. 1 Alanine (A) Glycine (G) Serine (S) Threonine (T) 2 AsparticGlutamic acid (D) acid (E) 3 Asparagine (N) Glutamine (Q) 4 Arginine (R)Histidine (H) Lysine (K) 5 Isoleucine (I) Leucine (L) Methionine (M)Valine (V) 6 Phenylala- Tyrosine (Y) Tryptophan (W) nine (F)

Still other examples of additional modifications are disclosed in thesections entitled “Modifications outside the Gla domain” and “Othermodifications outside the Gla domain”.

The term “nucleotide sequence” is intended to indicate a consecutivestretch of two or more nucleotide molecules. The nucleotide sequence maybe of genomic, cDNA, RNA, semisynthetic, synthetic origin, or anycombinations thereof.

The term “vector” refers to a plasmid or other nucleotide sequences thatare capable of replicating within a host cell or being integrated intothe host cell genome, and as such, are useful for performing differentfunctions in conjunction with compatible host cells (a vector-hostsystem) to facilitate the cloning of the nucleotide sequence, i.e. toproduce useful quantities of the sequence, to direct the expression ofthe gene product encoded by the sequence and to integrate the nucleotidesequence into the genome of the host cell. The vector will containdifferent components depending upon the function it is to perform.

“Cell”, “host cell”, “cell line” and “cell culture” are usedinterchangeably herein and all such terms should be understood toinclude progeny resulting from growth or culturing of a cell.

“Transformation” and “transfection” are used interchangeably to refer tothe process of introducing DNA into a cell.

“Operably linked” refers to the covalent joining of two or morenucleotide sequences, by means of enzymatic ligation or otherwise, in aconfiguration relative to one another such that the normal function ofthe sequences can be performed. Generally, “operably linked” means thatthe nucleotide sequences being linked are contiguous and, in the case ofa secretory leader, contiguous and in reading phase. Linking isaccomplished by ligation at convenient restriction sites. If such sitesdo not exist, then synthetic oligonucleotide adaptors or linkers areused, in conjunction with standard recombinant DNA methods.

In the context of the present invention the term “modification” or“amino acid modification” is intended to cover replacement of an aminoacid side chain, substitution of an amino acid residue, deletion of anamino acid residue or insertion of an amino acid residue.

The term “introduce” refers to introduction of an amino acid residue, inparticular by substitution of an existing amino acid residue, oralternatively by insertion of an additional amino acid residue.

The term “remove” refers to removal of an amino acid residue, inparticular by substitution of the amino acid residue to be removed byanother amino acid residue, or alternatively by deletion (withoutsubstitution) of the amino acid residue to be removed.

In the present context, the term “activity” should be understood as therelevant activity associated with the assay in which the activity isactually measured.

Thus, the term “amidolytic activity” is used to mean the activitymeasured in the “Amidolytic Assay” described herein. In order to exhibit“amidolytic activity” a variant of the invention, in its activated form,should have at least 10% of the amidolytic activity of rhFVIIa whenassayed in the “Amidolytic Assay” described herein. In a preferredembodiment of the invention the variant, in its activated form, has atleast 20% of the amidolytic activity of rhFVIIa, such as at least 30%,e.g. at least 40%, more preferably at least 50%, such as at least 60%,e.g. at least 70%, even more preferably at least 80%, such as at least90% of the amidolytic activity of rhFVIIa when assayed in the“Amidolytic Assay” described herein. In an interesting embodiment thevariant, in its activated form, has substantially the same amidolyticactivity as rhFVIIa, such as an amidolytic activity of 75-125% of theamidolytic activity of rhFVIIa.

The term “clotting activity” refers to the activity measured in the“Whole Blood Assay” described herein, i.e. the time needed to obtainclot formation. Thus, a lower clotting time corresponds to a higherclotting activity.

The term “increased clotting activity” is used to indicate that theclotting time of the polypeptide variant is statistically significantlydecreased relative to that generated by rhFVIIa or [P10Q+K32E]rhFVIIa asdetermined under comparable conditions and when measured in the “WholeBlood Assay” described herein.

In the present context, the term “activity” is also used in connectionwith the variants' capability of activating FX to FXa. This activity isalso denoted “FX activation activity” or “FXa generation activity” andmay be determined in the “TF-independent Factor X Activation Assay”described herein.

The term “increased FX activation activity” or “increased FXa generationactivity” is used to indicate that a variant of the invention, in itsactivated form, has a statistically significantly increased capabilityto activate FX to FXa as compared to a reference molecule, such asrhFVIIa or [P10Q+K32E]rhFVIIa. To what extent a variant of the invention(in its activated form) has an increased FX activation activity mayconveniently be determined in the “TF-independent Factor X ActivationAssay” described herein.

The term “immunogenicity” as used in connection with a given substanceis intended to indicate the ability of the substance to induce aresponse from the immune system. The immune response may be a cell orantibody mediated response (see, e.g., Roitt: Essential Immunology (10thEdition, Blackwell) for further definition of immunogenicity). Normally,reduced antibody reactivity will be an indication of reducedimmunogenicity. The immunogenicity may be determined by use of anysuitable method known in the art, e.g. in vivo or in vitro.

The term “functional in vivo half-life” is used in its normal meaning,i.e. the time at which 50% of the biological activity of the polypeptideis still present in the body/target organ, or the time at which theactivity of the polypeptide is 50% of the initial value.

As an alternative to determining functional in vivo half-life, “serumhalf-life” may be determined, i.e. the time at which 50% of thepolypeptide circulates in the plasma or bloodstream prior to beingcleared. Determination of serum half-life is often more simple thandetermining the functional in vivo half-life, and the magnitude of serumhalf-life is usually a good indication of the magnitude of functional invivo half-life. Alternative terms to serum half-life include “plasmahalf-life”, “circulating half-life”, “serum clearance”, “plasmaclearance” and “clearance half-life”. The polypeptide is cleared by theaction of one or more of the reticuloendothelial systems (RES), kidney,spleen or liver, by tissue factor, SEC receptor or other receptormediated elimination, or by specific or unspecific proteolysis.Normally, clearance depends on size (relative to the cutoff forglomerular filtration), charge, attached carbohydrate chains, and thepresence of cellular receptors for the protein. The functionality to beretained is normally selected from procoagulant, proteolytic or receptorbinding activity. The functional in vivo half-life and the serumhalf-life may be determined by any suitable method known in the art.

The term “increased” as used about the functional in vivo half-life orserum half-life is used to indicate that the relevant half-life of thepolypeptide variant is statistically significantly increased relative tothat of as reference molecule, such as rhFVIIa or [P10Q+K32E]rhFVIIa, asdetermined under comparable conditions (typically determined in anexperimental animal, such as rats, rabbits, pigs or monkeys).

The term “AUC_(iv)” or “Area Under the Curve when administeredintravenously” is used in its normal meaning, i.e. as the area under theactivity in serum-time curve, where the polypeptide variant has beenadministered intravenously, in particular when administeredintravenously in rats. Typically, the activity measured is the “clottingactivity” as defined above. Once the experimental activity-time pointshave been determined, the AUC_(iv) may conveniently be calculated by acomputer program, such as GraphPad Prism 3.01.

It will be understood that in order to make a direct comparison betweenthe AUC_(iv)-values of different molecules (e.g. between the variants ofthe invention and a reference molecule such as rhFVIIa or[P10Q+K32E]rhFVIIa) the same amount of activity should be administered.Consequently, the AUC_(iv)-values are typically normalized (i.e.corrected for differences in the injected dose) and expressed asAUC_(iv)/dose administered.

The term “reduced sensitivity to proteolytic degradation” is primarilyintended to mean that the polypeptide variant has reduced sensitivity toproteolytic degradation in comparison to hFVIIa, rhFVIIa or[P10Q+K32E]rhFVIIa as determined under comparable conditions.Preferably, the proteolytic degradation is reduced by at least 10% (e.g.by 10-25% or by 10-50%), such as at least 25% (e.g. by 25-50%, by 25-75%or by 25-100%), more preferably by at least 35%, such as at least 50%,(e.g. by 50-75% or by 50-100%) even more preferably by at least 60%,such as by at least 75% (e.g. by 75-100%) or even at least 90%.

The term “renal clearance” is used in its normal meaning to indicate anyclearance taking place by the kidneys, e.g. by glomerular filtration,tubular excretion or degradation in the tubular cells. Renal clearancedepends on physical characteristics of the polypeptide, including size(diameter), hydrodynamic volume, symmetry, shape/rigidity, and charge.Normally, a molecular weight of about 67 kDa is considered to be acut-off-value for renal clearance. Renal clearance may be established byany suitable assay, e.g. an established in vivo assay. Typically, renalclearance is determined by administering a labelled (e.g. radiolabelledor fluorescence labelled) polypeptide to a patient and measuring thelabel activity in urine collected from the patient. Reduced renalclearance is determined relative to a corresponding referencepolypeptide, e.g. rhFVIIa or [P10Q+K32E]rhFVIIa, under comparableconditions. Preferably, the renal clearance rate of the polypeptidevariant is reduced by at least 50%, preferably by at least 75%, and mostpreferably by at least 90% compared to rhFVIIa or [P10Q+K32E]rhFVIIa.

The terms “tissue factor binding site”, “active site region” and “ridgeof the active site binding cleft” are defined with reference to Example1.

The term “hydrophobic amino acid residue” includes the following aminoacid residues: Isoleucine (I), leucine (L), methionine (M), valine (V),phenylalanine (F), tyrosine (Y) and tryptophan (W).

The term “negatively charged amino acid residue” includes the followingamino acid residues: Aspartic acid (D) and glutamic acid (E).

The term “positively charged amino acid residue” includes the followingamino acid residues: Lysine (K), arginine (R) and histidine (H).

Variants of the Invention

The modifications performed in the Gla domain of the parent polypeptidepreferably provide the resulting molecule with an increased phospholipidmembrane binding affinity, an improved capability to activate FX to Fxa,and/or an increased clotting activity. The variants of the invention mayalso have a reduced tissue factor binding affinity and a reducedactivity when bound to tissue factor.

Without being limited by any particular theory, it is presently believedthat enhanced phospholipid membrane binding affinity results in a higherlocal concentration of the activated polypeptide variants in closeproximity to the other coagulation factors, particularly FX. Thus, therate of activation of FX to FXa will be higher, simply due to a highermolar ratio of the activated FVII variant to FX. The increasedactivation rate of FX then results in a higher amount of activethrombin, and thus a higher rate of cross-linking of fibrin.

Consequently, it is contemplated that medical treatment with apolypeptide variant according to the invention may provide advantagesover the currently available rhFVIIa compound (NovoSeven®), such as alower dose, increased efficacy and/or faster action.

Further, it is believed that tissue factor-independent variants, i.e.variants that have a reduced activity when bound to tissue factorcompared to wild-type human Factor VIIa, may offer certain safetyadvantages in terms of reduced risk of undesired blood clot formation(e.g. thrombosis or thromboembolism), in particular when used fortreatment of acute uncontrolled bleeding events such as trauma,including traumatic brain injury, or intracerebral haemorrhage.

Thus, in a highly preferred embodiment of the invention, the polypeptidevariant, in its activated form and when compared to a referencemolecule, such as rhFVIIa or [P10Q+K32E]rhFVIIa, has an increased FXactivation activity, in particular when assayed in a tissuefactor-independent assay, such as the “TF-independent Factor XActivation Assay” disclosed herein. More particularly, it is preferredthat the ratio between the FX activation activity of the polypeptidevariant, in its activated form, and the FX activation activity of areference molecule is at least 1.25 when assayed in the “TF-independentFactor X Activation Assay” disclosed herein. More preferably, this ratiois at least 1.5, such as at least 1.75, e.g. at least 2, even morepreferably at least 3, such as at least 4, most preferably at least 5.

When the reference molecule is rhFVIIa, the ratio between the FXactivation activity of the polypeptide variant, in its activated form,and the FX activation activity of rhFVIIa is preferably at least about5, typically at least about 10, when assayed in the “TF-independentFactor X Activation Assay” disclosed herein, such as at least about 15or 20.

In another highly preferred embodiment of the invention, the variants ofthe invention possess an increased clotting activity (i.e. a reducedclotting time) as compared to rhFVIIa or [P10Q+K32E]rhFVIIa. In apreferred embodiment of the invention the ratio between the time toreach clot formation for the variant (t_(variant)) and the time to reachclot formation for rhFVIIa (t_(wt)) or [P10Q+K32E]rhFVIIa(t_(P)10Q+K32E) is at the most 0.9 when assayed in the “Whole BloodAssay” described herein. More preferably this ratio is at the most 0.75,such as 0.7, even more preferably the ratio is at the most 0.6, and mostpreferably the ratio is at the most 0.5.

One or more of the above-mentioned properties may be achieved by themodifications described herein.

Variants of the Invention Comprising a Hydrophobic Amino Acid Residue inPosition 34

As indicated above, the present invention relates in a first aspect to aFVII or FVIIa polypeptide variant having an amino acid sequencecomprising 1-15 amino acid modifications relative to hFVII or hFVIIa(SEQ ID NO:1), wherein a hydrophobic amino acid residue has beenintroduced by substitution in position 34.

The hydrophobic amino acid residue to be introduced in position 34 maybe selected from the group consisting of I, L, M, V, F, Y and W,preferably I, L and V, in particular L.

In a preferred embodiment, the variant further comprises an amino acidsubstitution in position 10, in particular P10Q, and/or an amino acidsubstitution in position 32, in particular K32E. In a particularpreferred embodiment of the invention, the variant comprisessubstitutions in both of positions 10 and 32, such as P10Q+K32E.

Accordingly, in an interesting embodiment of the invention, the variantcomprises the substitutions P10Q+K32E+A34L.

In a particular interesting embodiment of the invention, the variantfurther comprises an insertion of at least one (typically one) aminoacid residue between position 3 and 4. It is preferred that the insertedamino acid residue is a hydrophobic amino acid residue. Most preferablythe insertion is A3AY. Accordingly, in a particular interestingembodiment of the invention, the variant comprises the modificationsA3AY+P10Q+K32E+A34L.

In addition to any of the above-mentioned modifications, the variant maycomprise a further substitution in position 33. Preferably, ahydrophobic amino acid residue is introduced by substitution in position33, in particular D33F.

The Gla domain may also contain modifications in other positions, inparticular in positions 8, 11 and 28, such as R28F or R28E. On the otherhand it should be understood that the Gla domain should not be modifiedto such an extent that the membrane binding properties are impaired.Accordingly, it is preferred that no modifications are made in theresidues that become γ-carboxylated, i.e. it is preferred that nomodifications are made in residues 6, 7, 14, 16, 19, 20, 25, 26, 29 and35. In a similar way, it is in general not preferred thatnon-polypeptide moieties, such as sugar moieties and/or PEG groups, areintroduced in the Gla domain. Consequently, it is preferred that nomodifications are made in the Gla domain that create an in vivoN-glycosylation site.

Finally, it will be understood that the modifications in the Gla domaindiscussed in this section may advantageously be combined with one ormore modifications in positions located outside the Gla domain (see thesections entitled “Modifications outside the Gla domain” and “Othermodifications outside the Gla domain” below).

Variants of the Invention Comprising an Amino Acid Substitution inPosition 36

As indicated above, the invention relates in a second aspect to a FVIIor FVIIa polypeptide variant having an amino acid sequence comprising1-15 amino acid modifications relative to hFVII or hFVIIa (SEQ ID NO:1),wherein said amino acid sequence comprises an amino acid substitution inposition 36.

Preferably, the amino acid residue top be introduced by substitution inposition 36 is a negatively charged amino acid residue, e.g. R36E orR36D, in particular R36E.

In a preferred embodiment, the variant further comprises an amino acidsubstitution in position 10, in particular P10Q, and/or an amino acidsubstitution in position 32, in particular K32E. In a particularpreferred embodiment of the invention, the variant comprisessubstitutions in both of positions 10 and 32, such as P10Q+K32E.

The variant of the invention may further contain a substitution inposition 38. It is preferred that a negatively charged amino acidresidue is introduced by substitution in position 38, e.g. K38E or K38D,in particular K38E.

Accordingly, interesting variants are those that comprise the followingsubstitutions P10Q+K32E+R36E or P10Q+K32E+R36E+K38E.

In a particular interesting embodiment, the variant further comprises anamino acid substitution in position 34 (i.e. the resulting variantcomprises substitutions in the following residues 10+32+34+36 or10+32+34+36+38). Preferably, a negatively charged amino acid residue isintroduced by substitution in position 34, e.g. A34E or A34D.

Specific examples of preferred variants are those that comprise thefollowing substitutions P10Q+K32E+A34E+R36E or P10Q+K32E+A34D+R36E+K38E.

In an interesting embodiment of the invention, the variant furthercomprises an insertion of at least one (typically one) amino acidresidue between position 3 and 4. It is preferred that the insertedamino acid residue is a hydrophobic amino acid residue. Most preferablythe insertion is A3AY.

In addition to any of the above-mentioned modifications, the variant maycomprise a further substitution in position 33. Preferably, ahydrophobic amino acid residue is introduced by substitution in position33, in particular D33F.

The Gla domain may also contain modifications in other positions, inparticular in positions 8, 11 and 28, such as R28F or R28E. On the otherhand it should be understood that the Gla domain should not be modifiedto such an extent that the membrane binding properties are impaired.Accordingly, it is preferred that no modifications are made in theresidues that become y-carboxylated, i.e. it is preferred that nomodifications are made in residues 6, 7, 14, 16, 19, 20, 25, 26, 29 and35. In a similar way, it is in general not preferred thatnon-polypeptide moieties, such as sugar moieties and/or PEG groups, areintroduced in the Gla domain. Consequently, it is preferred that nomodifications are made in the Gla domain that create an in vivoN-glycosylation site.

Finally, it will be understood that the modifications in the Gla domaindiscussed in this section may advantageously be combined with one ormore modifications in positions located outside the Gla domain (see thesections entitled “Modifications outside the Gla domain” and “Othermodifications outside the Gla domain” below).

Variants of the Invention Comprising Amino Acid Substitutions inPositions 74, 77 or 116

As indicated above, the present invention relates in a third aspect to aFVII or FVIIa polypeptide variant having an amino acid sequencecomprising 3-15 amino acid modifications relative to hFVII or hFVIIa(SEQ ID NO:1), wherein said amino acid sequence comprises an amino acidsubstitution in position 10, 32 and at least one further amino acidsubstitution in a position selected from the group consisting ofposition 74, 77 and 116.

In a preferred embodiment, the amino acid substitution in position 10 isP10Q and the amino acid substitution in position 32 is K32E

It is further preferred that the substitution in position 74, 77 or 116is selected from the group consisting of P74S, E77A and E116D.

In an interesting embodiment the variant further comprises an amino acidsubstitution in position 34. Preferably, a negatively charged amino acidresidue is introduced by substitution in position 34, e.g. A34E or A34D,in particular A34E.

In another interesting embodiment of the invention the variant furthercomprises an insertion of at least one (typically one) amino acidresidue between position 3 and 4. It is preferred that the insertedamino acid residue is a hydrophobic amino acid residue. Most preferablythe insertion is A3AY.

Thus, specific examples of interesting variants include variantscomprising the following modifications A3AY+P10Q+K32E+E116D,A3AY+P10Q+K32E+E77A and P10Q+K32E+A34E+P74S.

In addition to any of the above-mentioned modifications, the variant maycomprise a further substitution in position 33. Preferably, ahydrophobic amino acid residue is introduced by substitution in position33, in particular D33F.

The Gla domain may also contain modifications in other positions, inparticular in positions 8, 11 and 28, such as R28F or R28E. As explainedabove, the Gla domain should not be modified to such an extent that themembrane binding properties are impaired, i.e. preferably nomodifications are made in residues 6, 7, 14, 16, 19, 20, 25, 26, 29 and35, and it is preferred that an in vivo N-glycosylation site is notcreated in the Gla domain.

Finally, it will be understood that the modifications in the Gla domaindiscussed in this section may advantageously be combined with one ormore modifications in positions located outside the Gla domain (see thesections entitled “Modifications outside the Gla domain” and “OtherModifications Outside the Gla Domain” below).

Modifications Outside the Gla Domain

A circulating rhFVIIa half-life of 2.3 hours was reported in “SummaryBasis for Approval for NovoSeven®”, FDA reference number 96-0597.Relatively high doses and frequent administration are necessary to reachand sustain the desired therapeutic or prophylactic effect. As aconsequence, adequate dose regulation is difficult to obtain and theneed for frequent intravenous administration imposes restrictions on thepatient's way of living.

A molecule with a longer circulation half-life and/or increasedbioavailability (such as an increased Area Under the Curve as comparedto rhFVIIa when administered intravenously) would decrease the number ofnecessary administrations. Given the current need for frequentinjections and the potential for obtaining more optimal therapeuticFVIIa levels with concomitant enhanced therapeutic effect, there is aclear need for improved FVII- or FVIIa-like molecules.

Accordingly, a further object of the present invention is to provideimproved FVII or FVII molecules (FVII or FVIIa variants) with anincreased bioavailability (such as an increased Area Under the Curve ascompared to a reference molecule, such as rhFVIIa or [P10Q+K32E]rhFVIIa,when administered intravenously) and which are capable of activatingfactor X to factor Xa (without binding to tissue factor) moreefficiently than a reference molecule, such as rhFVIIa or[P10Q+K32E]rhFVIIa (thereby being able to treat uncontrolled bleadings,such as a trauma, or chronic conditions such as hemophilia moreefficiently).

Thus, interesting variants of the invention are those which, in theiractivated forms and when compared to a reference molecule, such asrhFVIIa or [P10Q+K32E]rhFVIIa, generate an increased Area Under theCurve when administered intravenously (AUC_(iv)). This can convenientlybe determined by intravenous administration in rats. More particularly,interesting variants of the present invention are those where the ratiobetween the AUC_(iv) of said variant, in its actvated form, and theAUC_(iv)of a reference molecule, such as rhFVIIa or [P10Q+K32E]rhFVIIa,is at least 1.25, such as at least 1.5, e.g. at least 1.75, morepreferably at least 2, such as at least 3, even more preferably at least4, such as at least 5, in particular when administered (intravenously)in rats.

This effect will often correspond to an increased functional in vivohalf-life and/or an increased serum half-life as compared to a referencemolecule, such as rhFVIIa or [P10Q+K32E]rhFVIIa. Accordingly, in anotherinteresting embodiment of the invention, the ratio between thefunctional in vivo half-life or the serum half-life for the variant, inits activated form, and the functional in vivo half-life or the serumhalf-life for a reference molecule, such as rhFVIIa or[P10Q+K32E]rhFVIIa, is at least 1.25. More preferably, the ratio betweenthe relevant half-life for the variant, in its activated form, and therelevant half-life for the reference molecule, such as rhFVIIa or[P10Q+K32E]rhFVIIa, is at least 1.5, such as at least 1.75, e.g. atleast 2, even more preferably at least 3, such as at least 4, e.g. atleast 5.

One way to increase the circulation half-life of a protein is to ensurethat renal clearance of the protein is reduced. This may be achieved byconjugating the protein to a chemical moiety which is capable ofconferring reduced renal clearance to the protein, e.g. polyethyleneglycol (PEG).

Furthermore, attachment of a chemical moiety to the protein orsubstitution of amino acids exposed to proteolysis may effectively blocka proteolytic enzyme from contact that otherwise leads to proteolyticdegradation of the protein.

As indicated above, instability due to proteolytic degradation is aknown problem in current rhFVIIa treatment. Proteolytic degradation isthus a major obstacle for obtaining a preparation in solution as opposedto a lyophilized product. The advantage of obtaining a stable solublepreparation lies in easier handling for the patient, and, in the case ofemergencies, quicker action, which potentially can become life saving.Attempts to prevent proteolytic degradation by site directed mutagenesisat major proteolytic sites have been disclosed in WO 88/10295.

WO 01/58935 discloses a number of suitable modifications leading to anincrease in AUC_(iv), functional in vivo half-life and/or serumhalf-life. The variants disclosed in WO 01/58935 are the result of agenerally new strategy for developing improved FVII or FVIIa molecules,which may also be used for the parent FVII or FVIIa polypeptide of thepresent invention.

More specifically, by removing and/or introducing an amino acid residuecomprising an attachment group for a non-polypeptide moiety in theparent FVII or FVIIa polypeptide it is possible to specifically adaptthe polypeptide so as to make the molecule more susceptible toconjugation to a non-polypeptide moiety of choice, to optimize theconjugation pattern (e.g. to ensure an optimal distribution and numberof non-polypeptide moieties on the surface of the FVII or FVIIapolypeptide variant and to ensure that only the attachment groupsintended to be conjugated is present in the molecule) and thereby obtaina new conjugate molecule which has amidolytic activity and in additionone or more improved properties as compared to rhFVIIa

In interesting embodiments of the present invention more than one aminoacid residue located outside the Gla domain is altered, e.g. thealteration embraces removal as well as introduction of amino acidresidues comprising an attachment group for the non-polypeptide moietyof choice. In addition to the removal and/or introduction of amino acidresidues the polypeptide variant may comprise other substitutions thatare not related to introduction and/or removal of amino acid residuescomprising an attachment group for the non-polypeptide moiety.

Also, the polypeptide variant may be attached to a serine proteinaseinhibitor to inhibit the catalytic site of the polypeptide variant.Alternatively, one or more of the amino acid residues present in thecatalytic site (S344, D242 and H193) may be mutated in order to renderthe resulting variant inactive. One example of such a mutation is S344A.

The amino acid residue comprising an attachment group for anon-polypeptide moiety, whether it be removed or introduced, is selectedon the basis of the nature of the non-polypeptide moiety of choice and,in most instances, on the basis of the method in which conjugationbetween the polypeptide variant and the non-polypeptide moiety is to beachieved. For instance, when the non-polypeptide moiety is a polymermolecule such as a polyethylene glycol or polyalkylene oxide derivedmolecule, amino acid residues comprising an attachment group may beselected from the group consisting of lysine, cysteine, aspartic acid,glutamic acid, histidine, and tyrosine, preferably lysine, cysteine,aspartic acid and glutamic acid, more preferably lysine and cysteine, inparticular cysteine.

Whenever an attachment group for a non-polypeptide moiety is to beintroduced into or removed from the parent polypeptide, the position ofthe amino acid residue to be modified is preferably located at thesurface of the parent FVII or FVIIa polypeptide, and more preferablyoccupied by an amino acid residue which has at least 25% of its sidechain exposed to the surface (as defined in Example 1 herein),preferably at least 50% of its side chain exposed to the surface (asdefined in Example 1 herein). Such positions have been identified on thebasis of an analysis of a 3D structure of the hFVII or hFVIIa moleculeas described in WO 01/58935.

Furthermore, the position to be modified is preferably selected from apart of the FVII or FVIIa molecule that is located outside the tissuefactor binding site, and/or outside the active site region, and/oroutside the ridge of the active site binding cleft. These sites/regionsare identified in Example 1 herein and in WO 01/58935.

In case of removal of an attachment group, the relevant amino acidresidue comprising such group and occupying a position as defined aboveis preferably substituted with a different amino acid residue that doesnot comprise an attachment group for the non-polypeptide moiety inquestion. Normally, the amino acid residue to be removed is one to whichconjugation is disadvantageous, e.g. an amino acid residue located at ornear a functional site of the polypeptide (since conjugation at such asite may result in inactivation or reduced activity of the resultingconjugate due to, e.g., impaired receptor recognition). In the presentcontext the term “functional site” is intended to indicate one or moreamino acid residues which is/are essential for or otherwise involved inthe function or performance of FVII or FVIIa. Such amino acid residuesare a part of the functional site. The functional site may be determinedby methods known in the art and is preferably identified by analysis ofa structure of the FVIIa-tissue factor complex (See Banner et al.,Nature 1996; 380:41-46).

In case of introduction of an attachment group, an amino acid residuecomprising such group is introduced into the relevant position,preferably by substitution of the amino acid residue occupying suchposition.

The exact number of attachment groups present and available forconjugation in the FVII or FVIIa polypeptide is dependent on the effectdesired to be achieved by the conjugation. The effect to be obtained is,e.g., dependent on the nature and degree of conjugation (e.g. theidentity of the non-polypeptide moiety, the number of non-polypeptidemoieties desirable or possible to conjugate to the polypeptide variant,where they should be conjugated or where conjugation should be avoided,etc.).

The total number of amino acid residues to be modified outside the Gladomain in the parent FVII or FVIIa polypeptide (as compared to the aminoacid sequence shown in SEQ ID NO:1) will typically not exceed 10.Preferably, the FVII or FVIIa variant comprises an amino acid sequencewhich differs in 1-10 amino acid residues from amino acid residues46-406 shown in SEQ ID NO:1, typically in 1-8 or in 2-8 amino acidresidues, e.g. in 1-5 or in 2-5 amino acid residues, such as in 1-4 orin 1-3 amino acid residues, e.g. in 1, 2 or 3 amino acid residues fromamino acid residues 46-406 shown in SEQ ID NO:1.

Analogously, the polypeptide variant of the invention may contain 1-10(additional) non-polypeptide moieties, typically 1-8 or 2-8 (additional)non-polypeptide moieties, preferably 1-5 or 2-5 (additional)non-polypeptide moieties, such as 1-4 or 1-3 (additional)non-polypeptide moieties, e.g. 1, 2 or 3 (additional) non-polypeptidemoieties. It will be understood that such additional non-polypeptidemoieties are covalently attached to an attachment group located outsidethe Gla domain.

Polypeptide Variants of the Invention Where the Non-Polypeptide Moietyis a Sugar Moiety

In a preferred embodiment of the invention, an attachment group for asugar moiety, such as a glycosylation site, in particular an in vivoglycosylation site, such as an in vivo N-glycosylation site, has beenintroduced and/or removed, preferably introduced, in a position locatedoutside the Gla domain.

When used in the present context, the term “naturally occurringglycosylation site” covers the glycosylation sites at postions N145,N322, S52 and S60. The term “naturally occurring in vivo O-glycosylationsite” includes the positions S52 and S60, whereas the term “naturallyoccurring in vivo N-glycosylation site” includes positions N145 andN322.

Thus, in a very interesting embodiment of the invention, thenon-polypeptide moiety is a sugar moiety and the introduced attachmentgroup is a glycosylation site, preferably an in vivo glycosylation site,such as an in vivo O-glycosylation site or an in vivo N-glycosylationsite, in particular an in vivo N-glycosylation site. Typically, 1-10glycosylation sites, in particular in vivo N-glycosylation sites, havebeen introduced, preferably 1-8, 1-6, 1-4 or 1-3 glycosylation sites, inparticular in vivo N-glycosylation sites, have been introduced in one ormore positionss located outside the Gla domain. For example 1, 2 or 3glycosylation sites, in particular in vivo N-glycosylation sites, mayhave been introduced outside the Gla domain, preferably by substitution.

It will be understood that in order to prepare a polypeptide variantwherein the polypeptide variant comprises one or more glycosylationsites, the polypeptide variant must be expressed in a host cell capableof attaching sugar (oligosaccharide) moieties at the glycosylationsite(s) or alternatively subjected to in vitro glycosylation. Examplesof glycosylating host cells are given in the section further belowentitled “Coupling to a sugar moiety ”.

Examples of positions wherein the glycosylation sites, in particular invivo N-glycosylation sites, may be introduced include amino acidresidues having at least 25% of their side chain exposed to the surface(as defined in Example 1 herein), such as at least 50% of the side chainexposed to the surface. The position is preferably selected from a partof the molecule that is located outside the tissue factor binding siteand/or the active site region and/or outside the ridge of the activesite cleft, as defined in Example 1 herein. It should be understood thatwhen the term “at least 25% (or at least 50%) of its side chain exposedto the surface” is used in connection with introduction of an in vivoN-glycosylation site this term refers to the surface accessibility ofthe amino acid side chain in the position where the sugar moiety isactually attached. In many cases it will be necessary to introduce aserine or a threonine residue in position +2 relative to the asparagineresidue to which the sugar moiety is actually attached, and thesepositions where the serine or threonine residues are introduced areallowed to be buried, i.e. to have less than 25% of their side chainsexposed to the surface.

Specific and preferred examples of such substitutions creating an invivo N-glycosylation site include a substitution selected from the groupconsisting of A51N, G58N, T106N, K109N, G124N, K143N+N145T, A175T,I205S, I205T, V253N, T267N, T267N+S269T, S314N+K316S, S314N+K316T,R315N+V317S, R315N+V317T, K316N+G318S, K316N+G318T, G318N, D334N andcombinations thereof. More preferably, the in vivo N-glycosylation siteis introduced by a substitution selected from the group consisting ofA51N, G58N, T106N, K109N, G124N, K143N+N145T, A175T, I205T, V253N,T267N+S269T, S314N+K316T, R315N+V317T, K316N+G318T, G318N, D334N andcombinations thereof. Even more preferably, the in vivo N-glycosylationsite is introduced by a substitution selected from the group consistingof T106N, A175T, I205T, V253N, T267N+S269T and combinations thereof, inparticular one, two or three of T106N, I205T and V253N.

In one embodiment, only one in vivo N-glycosylation site has beenintroduced by substitution. In another embodiment, two or more (such astwo) in vivo N-glycosylation sites have been introduced by substitution.Examples of preferred substitutions creating two in vivo N-glycosylationsites include substitutions selected from the group consisting ofA51N+G58N, A51N+T106N, A51N+K109N, A51N+G124N, A51N+K143N+N145T,A51N+A175T, A51N+I205T, A51N+V253N, A51N+T267N+S269T, A51N+S314N+K316T,A51N+R315N+V317T, A51N+K316N+G318T, A51N+G318N, A51N+D334N, G58N+T106N,G58N+K109N, G58N+G124N, G58N+K143N+N145T, G58N+A175T, G58N+I205T,G58N+V253N, G58N+T267N+S269T, G58N+S314N+K316T, G58N+R315N+V317T,G58N+K316N+G318T, G58N+G318N, G58N+D334N, T106N+K109N, T106N+G124N,T106N+K143N+N145T, T106N+A175T, T106N+I205T, T106N+V253N,T106N+T267N+S269T, T106N+S314N+K316T, T106N+R315N+V317T,T106N+K316N+G318T, T106N+G318N, T106N+D334N, K109N+G124N,K109N+K143N+N145T, K109N+A175T, K109N+I205T, K109N+V253N,K109N+T267N+S269T, K109N+S314N+K316T, K109N+R315N+V317T,K109N+K316N+G318T, K109N+G318N, K109N+D334N, G124N+K143N+N145T,G124N+A175T, G124N+I205T, G124N+V253N, G124N+T267N+S269T,G124N+S314N+K316T, G124N+R315N+V317T, G124N+K316N+G318T, G124N+G318N,G124N+D334N, K143N+N145T+A175T, K143N+N145T+I205T, K143N+N145T+V253N,K143N+N145T+T267N+S269T, K143N+N145T+S314N+K316T,K143N+N145T+R315N+V317T, K143N+N145T+K316N+G318T, K143N+N145T+G318N,K143N+N145T+D334N, A175T+I205T, A175T+V253N, A175T+T267N+S269T,A175T+S314N+K316T, A175T+R315N+V317T, A175T+K316N+G318T, A175T+G318N,A175T+D334N, I205T+V253N, I205T+T267N+S269T, I205T+S314N+K316T,I205T+R315N+V317T, I205T+K316N+G318T, I205T+G318N, I205T+D334N,V253N+T267N+S269T, V253N+S314N+K316T, V253N+R315N+V317T,V253N+K316N+G318T, V253N+G318N, V253N+D334N, T267N+S269T+S314N+K316T,T267N+S269T+R315N+V317T, T267N+S269T+K316N+G318T, T267N+S269T+G318N,T267N+S269T+D334N, S314N+K316T+R315N+V317T, S314N+K316T+G318N,S314N+K316T+D334N, R315N+V317T+K316N+G318T, R315N+V317T+G318N,R315N+V317T+D334N and G318N+D334N. More preferably, the substitutionsare selected from the group consisting of T106N+A175T, T106N+I205T,T106N+V253N, T106N+T267N+S269T, A175T+I205T, A175T+V253N,A175T+T267N+S269T, I205T+V253N, I205T+T267N+S269T and V253N+T267N+S269T,even more preferably from the group consisting of T106N+I205T,T106N+V253N and I205T+V253N.

In a further embodiment, three or more (such as three) in vivoN-glycosylation sites have been introduced by substitution. Examples ofpreferred substitutions creating three in vivo N-glycosylation sitesinclude substitutions selected from the group consisting ofI205T+V253N+T267N+S269T and T106N+I205T+V253N.

As discussed above, it is preferred that the in vivo N-glycosylationsite is introduced in a position which does not form part of the tissuefactor binding site, the active site region or the ridge of the activesite binding cleft as defined herein.

It will be understood that any of the modifications mentioned in theabove sections may be combined with each other, in addition to beingcombined with the above-described substitutions in position 34 and/or36, in particular A34E/L and/or R36E, and preferably in combination withthe above-described substitutions in position 10 and/or 32, inparticular P10Q and/or K32E. Among the above-identified modificationsfor introduction of an in vivo N-glycosylation site, preferredmodifications include one, two or three of T106N, I205T and V253N, inparticular two of these modifications, i.e. T106N+I205T, T106N+V253N orI205T+V253N.

Thus, in one preferred embodiment of the invention the FVII or FVIIavariant comprises the modifications P10Q+K32E+A34E+R36E+T106N+I205T.

In a further preferred embodiment the FVII or FVIIa variant comprisesthe modifications P10Q+K32E+A34E+R36E+T106N+V253N.

In a further preferred embodiment the FVII or FVIIa variant comprisesthe modifications P10Q+K32E+A34E+R36E+I205T+V253N.

In a further preferred embodiment the FVII or FVIIa variant comprisesthe modifications P10Q+K32E+A34L+T106N+1205T.

In a further preferred embodiment the FVII or FVIIa variant comprisesthe modifications P10Q+K32E+A34L+T106N+V253N.

In a further preferred embodiment the FVII or FVIIa variant comprisesthe modifications P10Q+K32E+A34L+I205T+V253N.

In a further preferred embodiment the FVII or FVIIa variant comprisesthe modifications P10Q+K32E+A34L+R36E+T106N+I205T.

In a further preferred embodiment the FVII or FVIIa variant comprisesthe modifications P10Q+K32E+A34L+R36E+T106N+V253N.

In a further preferred embodiment the FVII or FVIIa variant comprisesthe modifications P10Q+K32E+A34L+R36E+I205T+V253N.

As is also explained above, any one or more of these modifications mayin addition be combined with insertion of at least one amino acidresidue, typically a single amino acid residue, between position 3 and4, where the inserted residue is preferably a hydrophobic amino acidresidue. Most preferably the insertion is A3AY. Thus, in additionalpreferred embodiments of the invention the FVII or FVIIa variantcomprises modifications selected from:

A3AY+P10Q+K32E+A34E+R36E+T106N+I205T;

A3AY+P10Q+K32E+A34E+R36E+T106N+V253N;

A3AY+P10Q+K32E+A34E+R36E+I205T+V253N;

A3AY+P10Q+K32E+A34L+T106N+I205T;

A3AY+P10Q+K32E+A34L+T106N+V253N;

A3AY+P10Q+K32E+A34L+I205T+V253N;

A3AY+P10Q+K32E+A34L+R36E+T106N+I205T;

A3AY+P10Q+K32E+A34L+R36E+T106N+V253N;

A3AY+P10Q+K32E+A34L+R36E+I205T+V253N.

Other Modifications Outside the Gla Domain

In a further embodiment of the present invention, the FVII or FVIIavariant may, in addition to the modifications described in the sectionsabove, also contain mutations which are already known to increase theintrinsic activity of the polypeptide, for example those described in WO02/22776.

For example, the variant may comprise at least one modification in aposition selected from the group consisting of 157, 158, 296, 298, 305,334, 336, 337 and 374. Examples of preferred substitutions includesubstitutions selected from the group consisting of V158D, E296D, M298Q,L305V and K337A. More preferably, said substitutions are selected fromthe group consisting of V158D+E296D+M298Q+L305V+K337A,V158D+E296D+M298Q+K337A, V158D+E296D+M298Q+L305V, V158D+E296D+M298Q,M298Q, L305V+K337A, L305V and K337A.

In a further embodiment of the present invention, the FVII or FVIIavariant may, in addition to the modifications described in the sectionsabove, also contain other mutations, such as the substitution K341Qdisclosed by Neuenschwander et al, Biochemistry, 1995; 34:8701-8707.Other possible additional substitutions include D196K, D196N, G237L,G237GAA and combinations thereof.

Additional detailed information on conjugation of FVII and FVIIavariants to non-polypeptide moieties is found in WO 01/58935 and WO03/093465, to which reference is made and which are incorporated hereinby reference.

Methods of Preparing a Conjugated Variant of the Invention

In general, a conjugated variant according to the invention may beproduced by culturing an appropriate host cell under conditionsconducive for the expression of the variant polypeptide, and recoveringthe variant polypeptide, wherein a) the variant polypeptide comprises atleast one N— or O-glycosylation site and the host cell is an eukaryotichost cell capable of in vivo glycosylation, and/or b) the variantpolypeptide is subjected to conjugation to a non-polypeptide moiety invitro.

Conjugation to a Polymer Molecule

The polymer molecule to be coupled to the variant polypeptide may be anysuitable polymer molecule, such as a natural or synthetic homo-polymeror hetero-polymer, typically with a molecular weight in the range ofabout 300-100,000 Da, such as about 500-20,000 Da, more preferably inthe range of about 500-15,000 Da, even more preferably in the range ofabout 2-12 kDa, such as in the range of about 3-10 kDa. When the term“about” is used herein in connection with a certain molecular weight,the word “about” indicates an approximate average molecular weight andreflects the fact that there will normally be a certain molecular weightdistribution in a given polymer preparation.

Examples of homo-polymers include a polyol (i.e. poly-OH), a polyamine(i.e. poly-NH₂) and a polycarboxylic acid (i.e. poly-COOH). Ahetero-polymer is a polymer comprising different coupling groups, suchas a hydroxyl group and an amine group.

Examples of suitable polymer molecules include polymer moleculesselected from the group consisting of polyalkylene oxide (PAO),including polyalkylene glycol (PAG), such as polyethylene glycol (PEG)and polypropylene glycol (PPG), branched PEGs, poly-vinyl alcohol (PVA),poly-carboxylate, poly-(vinylpyrolidone), polyethylene-co-maleic acidanhydride, polystyrene-co-maleic acid anhydride, dextran, includingcarboxymethyl-dextran, or any other biopolymer suitable for reducingimmunogenicity and/or increasing functional in vivo half-life and/orserum half-life. Another example of a polymer molecule is human albuminor another abundant plasma protein. Generally, polyalkyleneglycol-derived polymers are biocompatible, non-toxic, non-antigenic,non-immunogenic, water soluble, and are easily excreted from livingorganisms.

PEG is the preferred polymer molecule, since it has only few reactivegroups capable of cross-linking compared to, e.g., polysaccharides suchas dextran. In particular, monofunctional PEG, e.g. methoxypolyethyleneglycol (mPEG), is of interest since its coupling chemistry is relativelysimple (only one reactive group is available for conjugating withattachment groups on the polypeptide). Consequently, as the risk ofcross-linking is eliminated, the resulting conjugated variants are morehomogeneous and the reaction of the polymer molecules with the variantpolypeptide is easier to control.

To effect covalent attachment of the polymer molecule(s) to the variantpolypeptide, the hydroxyl end groups of the polymer molecule must beprovided in activated form, i.e. with reactive functional groups(examples of which include primary amino groups, hydrazide (HZ), thiol,succinate (SUC), succinimidyl succinate (SS), succinimidyl succinamide(SSA), succinimidyl propionate (SPA), succinimidyl butyrate (SBA),succinimidyl carboxymethylate (SCM), benzotriazole carbonate (BTC),N-hydroxysuccinimide (NHS), aldehyde, nitrophenylcarbonate (NPC), andtresylate (TRES)). Suitable activated polymer molecules are commerciallyavailable, e.g. from Nektar Therapeutics, Huntsville, Ala., USA, or fromPolyMASC Pharmaceuticals plc, UK.

Specific examples of activated linear or branched polymer molecules foruse in the present invention are described in the Nektar MoleculeEngineering Catalog 2003 (Nektar Therapeutics), incorporated herein byreference.

Specific examples of activated PEG polymers include the following linearPEGs: NHS-PEG (e.g. SPA-PEG, SSPA-PEG, SBA-PEG, SS-PEG, SSA-PEG, SC-PEG,SG-PEG, and SCM-PEG), and NOR-PEG, BTC-PEG, EPOX-PEG, NCO-PEG, NPC-PEG,CDI-PEG, ALD-PEG, TRES-PEG, VS-PEG, IODO-PEG, and MAL-PEG, and branchedPEGs such as PEG2-NHS and those disclosed in U.S. Pat. No. 5,932,462 andU.S. Pat. No. 5,643,575, both of which are incorporated herein byreference. Additional publications disclosing useful polymer molecules,PEGylation chemistries and conjugation methods are listed in WO 01/58935and WO 03/093465.

Specific examples of activated PEG polymers particularly preferred forcoupling to cysteine residues, include the following linear PEGs:vinylsulfone-PEG (VS-PEG), preferably vinylsulfone-mPEG (VS-mPEG);maleimide-PEG (MAL-PEG), preferably maleimide-mPEG (MAL-mPEG) andorthopyridyl-disulfide-PEG (OPSS-PEG), preferablyorthopyridyl-disulfide-mPEG (OPSS-mPEG). Typically, such PEG or mPEGpolymers will have a size of about 5 kDa, about 10 kD, about 12 kDa orabout 20 kDa.

The skilled person will be aware that the activation method and/orconjugation chemistry to be used depends on the attachment group(s) ofthe variant polypeptide (examples of which are given further above), aswell as the functional groups of the polymer (e.g. being amine,hydroxyl, carboxyl, aldehyde, sulfydryl, succinimidyl, maleimide,vinysulfone or haloacetate). The PEGylation may be directed towardsconjugation to all available attachment groups on the variantpolypeptide (i.e. such attachment groups that are exposed at the surfaceof the polypeptide) or may be directed towards one or more specificattachment groups, e.g. the N-terminal amino group as described in U.S.Pat. No. 5,985,265 or to cysteine residues. Furthermore, the conjugationmay be achieved in one step or in a stepwise manner (e.g. as describedin WO 99/55377).

For PEGylation to cysteine residues (see above) the FVII or FVIIavariant is usually treated with a reducing agent, such as dithiothreitol(DDT) prior to PEGylation. The reducing agent is subsequently removed byany conventional method, such as by desalting. Conjugation of PEG to acysteine residue typically takes place in a suitable buffer at pH 6-9 attemperatures varying from 4° C. to 25° C. for periods up to 16 hours.

It will be understood that the PEGylation is designed so as to producethe optimal molecule with respect to the number of PEG moleculesattached, the size and form of such molecules (e.g. whether they arelinear or branched), and the attachment site(s) in the variantpolypeptide. The molecular weight of the polymer to be used may e.g. bechosen on the basis of the desired effect to be achieved.

In connection with conjugation to only a single attachment group on theprotein (e.g. the N-terminal amino group), it may be advantageous thatthe polymer molecule, which may be linear or branched, has a highmolecular weight, preferably about 10-25 kDa, such as about 15-25 kDa,e.g. about 20 kDa.

Normally, the polymer conjugation is performed under conditions aimed atreacting as many of the available polymer attachment groups as possiblewith polymer molecules. This is achieved by means of a suitable molarexcess of the polymer relative to the polypeptide. Typically, the molarratios of activated polymer molecules to polypeptide are up to about1000-1, such as up to about 200-1, or up to about 100-1. In some casesthe ration may be somewhat lower, however, such as up to about 50-1,10-1, 5-1, 2-1 or 1-1 in order to obtain optimal reaction.

It is also contemplated according to the invention to couple the polymermolecules to the polypeptide through a linker. Suitable linkers are wellknown to the skilled person; see also WO 01/58935.

Subsequent to the conjugation, residual activated polymer molecules areblocked according to methods known in the art, e.g. by addition ofprimary amine to the reaction mixture, and the resulting inactivatedpolymer molecules are removed by a suitable method.

It will be understood that depending on the circumstances, e.g. theamino acid sequence of the variant polypeptide, the nature of theactivated PEG compound being used and the specific PEGylationconditions, including the molar ratio of PEG to polypeptide, varyingdegrees of PEGylation may be obtained, with a higher degree ofPEGylation generally being obtained with a higher ratio of PEG tovariant polypeptide. The PEGylated variant polypeptides resulting fromany given PEGylation process will, however, normally comprise astochastic distribution of conjugated polypeptide variants havingslightly different degrees of PEGylation.

Coupling to a Sugar Moiety

In order to achieve in vivo glycosylation of a FVII molecule comprisingone or more glycosylation sites the nucleotide sequence encoding thevariant polypeptide must be inserted in a glycosylating, eucaryoticexpression host. The expression host cell may be selected from fungal(filamentous fungal or yeast), insect or animal cells or from transgenicplant cells. In one embodiment the host cell is a mammalian cell, suchas a CHO cell, BHK or HEK, e.g. HEK 293, cell, or an insect cell, suchas an SF9 cell, or a yeast cell, e.g. Saccharomyces cerevisiae or Pichiapastoris, or any of the host cells mentioned hereinafter.

Covalent in vitro coupling of sugar moieties (such as dextran) to aminoacid residues of the variant polypeptide may also be used, e.g. asdescribed, for example in WO 87/05330 and in Aplin et al., CRC Crit Rev.Biochem, pp. 259-306, 1981. See also WO 03/093465 for furtherinformation on in vitro glycosylation of variants of FVII or FVIIa.

Attachment of Serine Protease Inhibitor

Attachment of a serine protease inhibitor can be performed in accordancewith the method described in WO 96/12800.

Methods of Preparing a Polypeptide Variant of the Invention

The polypeptide variant of the present invention, optionally inglycosylated form, may be produced by any suitable method known in theart. Such methods include constructing a nucleotide sequence encodingthe polypeptide variant and expressing the sequence in a suitabletransformed or transfected host. Preferably, the host cell is agamma-carboxylating host cell such as a mammalian cell. However,polypeptide variants of the invention may be produced, albeit lessefficiently, by chemical synthesis or a combination of chemicalsynthesis or a combination of chemical synthesis and recombinant DNAtechnology.

A nucleotide sequence encoding a polypeptide of the invention may beconstructed by isolating or synthesizing a nucleotide sequence encodingthe parent FVII, such as hFVII with the amino acid sequence shown in SEQID NO:1 and then changing the nucleotide sequence so as to effectintroduction (i.e. insertion or substitution) or removal (i.e. deletionor substitution) of the relevant amino acid residue(s).

The nucleotide sequence is conveniently modified by site-directedmutagenesis in accordance with conventional methods. Alternatively, thenucleotide sequence is prepared by chemical synthesis, e.g. by using anoligonucleotide synthesizer, wherein oligonucleotides are designed basedon the amino acid sequence of the desired polypeptide, and preferablyselecting those codons that are favored in the host cell in which therecombinant polypeptide will be produced. For example, several smalloligonucleotides coding for portions of the desired polypeptide may besynthesized and assembled by PCR (polymerase chain reaction), ligationor ligation chain reaction (LCR) (Barany, Proc Natl Acad Sci USA88:189-193, 1991). The individual oligonucleotides typically contain 5′or 3′ overhangs for complementary assembly.

Once assembled (by synthesis, site-directed mutagenesis or anothermethod), the nucleotide sequence encoding the polypeptide is insertedinto a recombinant vector and operably linked to control sequencesnecessary for expression of the FVII in the desired transformed hostcell.

Persons skilled in the art will be capable of selecting suitablevectors, expression control sequences and hosts for expressing thepolypeptide. The recombinant vector may be an autonomously replicatingvector, i.e. a vector, which exists as an extrachromosomal entity, thereplication of which is independent of chromosomal replication, e.g. aplasmid. Alternatively, the vector is one which, when introduced into ahost cell, is integrated into the host cell genome and replicatedtogether with the chromosome(s) into which it has been integrated.

The vector is preferably an expression vector, in which the nucleotidesequence encoding the polypeptide variant of the invention is operablylinked to additional segments required for transcription of thenucleotide sequence. The vector is typically derived from plasmid orviral DNA. A number of suitable expression vectors for expression in thehost cells mentioned herein are commercially available or described inthe literature. Detailed information on suitable vectors for expressingFVII may be found in WO 01/58935, incorporated by reference.

The term “control sequences” is defined herein to include all componentswhich are necessary or advantageous for the expression of thepolypeptide variant of the invention. Each control sequence may benative or foreign to the nucleic acid sequence encoding the polypeptidevariant. Such control sequences include, but are not limited to, aleader sequence, polyadenylation sequence, propeptide sequence,promoter, enhancer or upstream activating sequence, signal peptidesequence, and transcription terminator. At a minimum, the controlsequences include a promoter.

A wide variety of expression control sequences may be used in thepresent invention, e.g. any of the control sequences disclosed in WO01/58935, incorporated by reference.

The nucleotide sequence of the invention encoding a polypeptide variant,whether prepared by site-directed mutagenesis, synthesis, PCR or othermethods, may optionally include a nucleotide sequence that encode asignal peptide. The signal peptide is present when the polypeptidevariant is to be secreted from the cells in which it is expressed. Suchsignal peptide, if present, should be one recognized by the cell chosenfor expression of the polypeptide variant. The signal peptide may behomologous (i.e. normally associated with hFVII) or heterologous (i.e.originating from another source than hFVII) to the polypeptide or may behomologous or heterologous to the host cell, i.e. a signal peptidenormally expressed from the host cell or one which is not normallyexpressed from the host cell. For further information on suitable signalpeptides, see WO 01/58935.

Any suitable host may be used to produce the polypeptide variant,including bacteria (although not particularly preferred), fungi(including yeasts), plant, insect, mammal, or other appropriate animalcells or cell lines, as well as transgenic animals or plants. Mammaliancells are preferred. Examples of bacterial host cells includegram-positive bacteria such as strains of Bacillus, e.g. B. brevis or B.subtilis, Pseudomonas or Streptomyces, or gram-negative bacteria, suchas strains of E. coli. Examples of suitable filamentous fungal hostcells include strains of Aspergillus, e.g. A. oryzae, A. niger, or A.nidulans, Fusarium or Trichoderma. Examples of suitable yeast host cellsinclude strains of Saccharomyces, e.g. S. cerevisiae,Schizosaccharomyces, Klyveromyces, Pichia, such as P. pastoris or P.methanolica, Hansenula, such as H. Polymorpha or Yarrowia. Examples ofsuitable insect host cells include a Lepidoptora cell line, such asSpodoptera frugiperda (Sf9 or Sf21) or Trichoplusioa ni cells (HighFive) (U.S. Pat. No. 5,077,214). Examples of suitable mammalian hostcells include Chinese hamster ovary (CHO) cell lines, (e.g. CHO—K1; ATCCCCL-61), Green Monkey cell lines (COS) (e.g. COS 1 (ATCC CRL-1650), COS7 (ATCC CRL-1651)); mouse cells (e.g. NS/O), Baby Hamster Kidney (BHK)cell lines (e.g. ATCC CRL-1632 or ATCC CCL-10), and human cells (e.g.HEK 293 (ATCC CRL-1573)). Additional suitable cell lines are known inthe art and available from public depositories such as the American TypeCulture Collection, Rockville, Md. Also, mammalian cells, such as a CHOcell, may be modified to express sialyltransferase, e.g.1,6-sialyltransferase, e.g. as described in U.S. Pat. No. 5,047,335, inorder to provide improved glycosylation of the polypeptide variant.

In order to increase secretion it may be of particular interest toproduce the polypeptide variant of the invention together with anendoprotease, in particular a PACE (paired basic amino acid convertingenzyme) (e.g. as described in U.S. Pat. No. 5,986,079), such as a Kex2endoprotease (e.g. as described in WO 00/28065).

Methods for introducing exogeneous DNA into the above cell types, aswell as other information regarding expression, production andpurification of FVII variants, is found in WO 01/58935, incorporatedherein by reference.

Pharmaceutical Composition of the Invention and its Use

In a further aspect, the present invention relates to a composition, inparticular to a pharmaceutical composition, comprising a polypeptidevariant of the invention and a pharmaceutically acceptable carrier orexcipient.

The polypeptide variant or the pharmaceutical composition according tothe invention may be used as a medicament.

Due to the improved properties mentioned above, the polypeptide variantsof the invention, or the pharmaceutical composition of the invention,are particular useful for the treatment of uncontrollable bleedingevents in trauma patients, thrombocytopenic patients, patients inanticoagulant treatment, and cirrhosis patients with variceal bleeding,or other upper gastrointestinal bleedings, in patients undergoingorthotopic liver transplantation or liver resection (allowing fortransfusion free surgery), or in hemophilia patients.

Trauma is defined as an injury to living tissue caused by an extrinsicagent. It is the 4^(th) leading cause of death in the US and places alarge financial burden on the economy.

Trauma is classified as either blunt or penetrative. Blunt traumaresults in internal compression, organ damage and internal haemorrhagewhereas penetrative trauma (as the consequence of an agent penetratingthe body and destroying tissue, vessels and organs) results in externalhaemorrhage.

Trauma may be caused by numerous events, e.g. traffic accidents, gunshotwounds, falls, machinery accidents, and stab wounds.

Cirrhosis of the liver may be caused by direct liver injury, includingchronic alcoholism, chronic viral hepatitis (types B, C, and D), andautoimmune hepatitis as well as by indirect injury by way of bile ductdamage, including primary biliary cirrhosis, primary sclerosingcholangitis and biliary atresia. Less common causes of cirrhosis includedirect liver injury from inherited disease such as cystic fibrosis,alpha-1-antitrypsin deficiency, hemochromatosis, Wilson's disease,galactosemia, and glycogen storage disease. Transplantation is the keyintervention for treating late stage cirrhotic patients

Thus, in a further aspect the present invention relates to a polypeptidevariant of the invention for the manufacture of a medicament for thetreatment of diseases or disorder wherein clot formation is desirable. Astill further aspect of the present invention relates to a method fortreating a mammal having a disease or disorder wherein clot formation isdesirable, comprising administering to a mammal in need thereof aneffective amount of the polypeptide variant or the pharmaceuticalcomposition of the invention.

Examples of diseases/disorders wherein increased clot formation isdesirable include, but is not limited to, hemorrhages, including brainhemorrhages, as well as patient with severe uncontrolled bleedings, suchas trauma. Further examples include patients undergoing livingtransplantations, patients undergoing resection and patients withvariceal bleeding. Another widespread disease/disorder in which it iscontemplated that the polypeptides of the invention will be useful forincreased clot formation is hemophilia, e.g. von Willebrand disease,hemophilia A, hemophilia B or hemophilia C.

As mentioned above, one particular aspect of the invention relates touse of the polypeptide variants of the invention for the treatment ofintracerebral haemorrhage (ICH) or traumatic brain injury (TBI).

Among US and European populations, an estimated 10-15% of all strokecases are caused by intracerebral haemorrhage, also known as brainhaemorrhage, intracranial haemorrhage or haemorrhagic stroke, while thefigure for Asian populations is estimated to be 20-30%. ICH is the mostdeadly form of stroke, for which there currently is no proven effectivetreatment. In addition to high short-term mortality rates, ICH alsoresults in very high rates of severe mental and physical disabilityamong survivors.

ICH can be distinguished from other types of stroke using a CT scan orMRI, after which treatment may be initiated, although until now theavailable treatment options have only been symptomatic and largelyineffective. If initiated sufficiently early, however, e.g. within about3-4 hours of the onset of the haemorrhagic stroke, it is contemplatedthat treatment with the polypeptide variants of the invention may resultin significant improvements in terms of increase survival rates and/ordecreased disability rates. In particular, it is contemplated that anincreased TF-independent activity, optionally with a reducedTF-dependent activity, obtained by use of the polypeptide variants ofthe invention, may be advantageous over rhFVIIa (NovoSeven®) by reducingor eliminating the risk of thromboembolic events.

Traumatic brain injury is another public health problem with highmortality rates and a high frequency of long-term disability. In theUnited States alone, there are an estimated 500,000 cases of TBI eachyear, and also here there is a lack of proven, effective treatments. Theavailable data on clinical trials in head injury is reviewed by Narayanet al., J. Neurotrama (2002) 19(5):503-557.

One embodiment of this aspect of the invention thus relates to a methodfor treating ICH or TBI, comprising administering to a patient in needthereof an effective amount of a polypeptide variant of the invention asotherwise described above. Another embodiment of this aspect of theinvention relates to use of a polypeptide of the invention for themanufacture of a medicament for the treatment of ICH or TBI.

In one embodiment, this aspect of the invention may generally be definedas a method for treating intracerebral haemorrhage or traumatic braininjury, comprising administering to a patient in need thereof aneffective amount of a Factor VII (FVII) or Factor VIIa (FVIIa)polypeptide variant having an amino acid sequence comprising 1-15 aminoacid modifications relative to human Factor VII (hFVII) or human FactorVIIa (hFVIIa) with the amino acid sequence shown in SEQ ID NO:1, whereinthe FX activation activity of the polypeptide variant, in its activatedform, is greater than the FX activation activity of rhFVIIa when assayedin the “TF-independent Factor X Activation Assay” disclosed herein. Inparticular, the ratio between the FX activation activity of thepolypeptide variant, in its activated form, and the FX activationactivity of rhFVIIa is preferably at least about 5, more preferably atleast about 10, such as at least about 15.

Further, in addition to use of the variants described above, i.e.comprising at least one modification selected from (a) introduction of ahydrophobic amino acid residue by substitution in position 34, and (b)an amino acid substitution in position 36, for the treatment of ICH orTBI, it is also contemplated that certain other FVIIa variants havingincreased efficacy and preferably a decreased TF-dependent activity maybe advantageous over rhFVIIa for these indications. These variants havean amino acid sequence comprising 1-15 amino acid modifications relativeto human Factor VII (hFVII) or human Factor VIIa (hFVIIa) with the aminoacid sequence shown in SEQ ID NO:1, and comprise an amino acidsubstitution in position 10 and/or 32, and optionally at least onefurther substitution to introduce an in vivo N-glycosylation site.Preferably, the substitution in position 10 is P10Q and the substitutionin position 32 is K32E. More preferably, the variant includes bothsubstitutions P10Q and K32E, and optionally one or more additionalsubstitutions to introduce at least one in vivo N-glycosylation site,e.g. one, two or three glycosylation sites, preferably two glycosylationsites, selected from A51N, G58N, T106N, K109N, G124N, K143N+N145T,A175T, I205S, I205T, V253N, T267N, T267N+S269T, S314N+K316S,S314N+K316T, R315N+V317S, R315N+V317T, K316N+G318S, K316N+G318T, G318Nand D334N. Still more preferably, the variant includes the substitutionsP10Q and K32E as well as two substitutions selected from T106N, I205S/Tand V253N, most preferably P10Q+K32E+T106N+V253N. The polypeptidevariants of the invention are administered to patients in atherapeutically effective dose, normally one approximately parallelingthat employed in therapy with rFVII such as NovoSeven®. By“therapeutically effective dose” herein is meant a dose that issufficient to produce the desired effects in relation to the conditionfor which it is administered. The exact dose will depend on thecircumstances, and will be ascertainable by one skilled in the art usingknown techniques. Normally, the dose should be capable of preventing orlessening the severity or spread of the condition or indication beingtreated. It will be apparent to those of skill in the art that aneffective amount of a polypeptide variant or composition of theinvention depends, inter alia, upon the disease, the dose, theadministration schedule, whether the polypeptide variant or compositionis administered alone or in conjunction with other therapeutic agents,the plasma half-life of the compositions, and the general health of thepatient.

It is contemplated that a suitable dose of the variants of the inventionfor treatment of ICH, or TBI or other trauma, will be in the range ofabout 20-300 μg protein per kg body weight, e.g. about 30-250 μg/kg,such as about 40-200 μg/kg, e.g. about 60-150 μg/kg. For ICH, only asingle dose of the of the variants of the invention will generally beindicated, while for TBI or other forms of trauma one or more additionaldoses may in certain cases be given as needed. Similar dose ranges arealso applicable to hemophilia.

The polypeptide variant of the invention is preferably administered in acomposition including a pharmaceutically acceptable carrier orexcipient. “Pharmaceutically acceptable” means a carrier or excipientthat does not cause any untoward effects in patients to whom it isadministered. Such pharmaceutically acceptable carriers and excipientsas well as suitable pharmaceutical formulation methods are well known inthe art (see, for example, Remington's Pharmaceutical Sciences, 18thedition, A. R. Gennaro, Ed., Mack Publishing Company [1990];Pharmaceutical Formulation Development of Peptides and Proteins, S.Frokjaer and L. Hovgaard, Eds., Taylor & Francis [2000]; and Handbook ofPharmaceutical Excipients, 3rd edition, A. Kibbe, Ed., PharmaceuticalPress [2000]).

The polypeptide variant of the invention can be used “as is” and/or in asalt form thereof. Suitable salts include, but are not limited to, saltswith alkali metals or alkaline earth metals, such as sodium, potassium,calcium and magnesium, as well as e.g. zinc salts. These salts orcomplexes may by present as a crystalline and/or amorphous structure.

The pharmaceutical composition of the invention may be administeredalone or in conjunction with other therapeutic agents. These agents maybe incorporated as part of the same pharmaceutical composition or may beadministered separately from the polypeptide variant of the invention,either concurrently or in accordance with another treatment schedule. Inaddition, the polypeptide variant or pharmaceutical composition of theinvention may be used as an adjuvant to other therapies.

A “patient” for the purposes of the present invention includes bothhumans and other mammals. Thus, the methods are applicable to both humantherapy and veterinary applications, in particular to human therapy.

The pharmaceutical composition comprising the polypeptide variant of theinvention may be formulated in a variety of forms, e.g. as a liquid,gel, lyophilized, or as a compressed solid. The preferred form willdepend upon the particular indication being treated and will be apparentto one skilled in the art.

In particular, the pharmaceutical composition comprising the polypeptidevariant of the invention may be formulated in lyophilised or stablesoluble form. The polypeptide variant may be lyophilised by a variety ofprocedures known in the art. The polypeptide variant may be in a stablesoluble form by the removal or shielding of proteolytic degradationsites as described herein. The advantage of obtaining a stable solublepreparation lies in easier handling for the patient and, in the case ofemergencies, quicker action, which potentially can become life saving.The preferred form will depend upon the particular indication beingtreated and will be apparent to one of skill in the art.

The administration of the formulations of the present invention can beperformed in a variety of ways, including, but not limited to, orally,subcutaneously, intravenously, intracerebrally, intranasally,transdermally, intraperitoneally, intramuscularly, intrapulmonary,vaginally, rectally, intraocularly, or in any other acceptable manner.The formulations can be administered continuously by infusion, althoughbolus injection is acceptable, using techniques well known in the art,such as pumps or implantation. In some instances the formulations may bedirectly applied as a solution or spray.

Parentals

A preferred example of a pharmaceutical composition is a solution, inparticular an aqueous solution, designed for parenteral administration.Although in many cases pharmaceutical solution formulations are providedin liquid form, appropriate for immediate use, such parenteralformulations may also be provided in frozen or in lyophilized form. Inthe former case, the composition must be thawed prior to use. The latterform is often used to enhance the stability of the active compoundcontained in the composition under a wider variety of storageconditions, as it is recognized by those skilled in the art thatlyophilized preparations are generally more stable than their liquidcounterparts. Such lyophilized preparations are reconstituted prior touse by the addition of one or more suitable pharmaceutically acceptablediluents such as sterile water for injection or sterile physiologicalsaline solution.

In case of parenterals, they are prepared for storage as lyophilizedformulations or aqueous solutions by mixing, as appropriate, thepolypeptide variant having the desired degree of purity with one or morepharmaceutically acceptable carriers, excipients or stabilizerstypically employed in the art (all of which are termed “excipients”),for example buffering agents, stabilizing agents, preservatives,isotonifiers, non-ionic surfactants or detergents, antioxidants, and/orother miscellaneous additives such as bulking agents or fillers,chelating agents, antioxidants and cosolvents.

Detailed information on parental formulations suitable foradministration of FVII variants, as well as sustained releasepreparations, is found in WO 01/58935 and WO 03/093465, incorporatedherein by reference.

The invention is further described by the following non-limitingexamples.

Materials and Methods

Active Site Region

The active site region is defined as any residues having at least oneatom within 10 Å of any atom in the catalytic triad (residues H193,D242, S344).

Measurement of Reduced Sensitivity to Proteolytic Degradation

Proteolytic degradation can be measured using the assay described inU.S. Pat. No. 5,580,560, Example 5, where proteolysis isautoproteolysis.

Furthermore, reduced proteolysis can be tested in an in vivo model usingradiolabelled samples and comparing proteolysis of rhFVIIa and thepolypeptide variant of the invention by withdrawing blood samples andsubjecting these to SDS-PAGE and autoradiography.

Irrespective of the assay used for determining proteolytic degradation,“reduced proteolytic degradation” is intended to mean a measurablereduction in cleavage compared to that obtained by rhFVIIa as measuredby gel scanning of Coomassie stained SDS-PAGE gels, HPLC or as measuredby conserved catalytic activity in comparison to wild type using thetissue factor independent activity assay decribed below.

Determination of the Molecular Weight of Polypeptide Variants

The molecular weight of polypeptide variants is determined by eitherSDS-PAGE, gel filtration, Western Blots, matrix assisted laserdesorption mass spectrometry or equilibrium centrifugation, e.g.SDS-PAGE according to Laemmli, U.K., Nature Vol 227 (1970), pp. 680-85.

Determination of Phospholipid Membrane Binding Affinity

Phospholipid membrane binding affinity may be determined as described inNelsestuen et al., Biochemistry, 1977; 30;10819-10824 or as described inExample 1 in U.S. Pat. No. 6,017,882.

TF-Independent Factor X Activation Assay

This assay has been described in detail on page 39826 in Nelsestuen etal., J Biol Chem, 2001; 276:39825-39831.

Briefly, the molecule to be assayed (either hFVIIa, rhFVIIa or thepolypeptide variant of the invention in its activated form) is mixedwith a source of phospholipid (preferably phosphatidylcholine andphosphatidylserine in a ratio of 8:2) and relipidated Factor X in Trisbuffer containing BSA. After a specified incubation time the reaction isstopped by addition of excess EDTA. The concentration of factor Xa isthen measured from absorbance change at 405 nm after addition of achromogenic substrate (S-2222, Chromogenix). After correction forbackground the tissue factor independent activity of rhFVIIa (a_(wt)) isdetermined as the absorbance change after 10 minutes and the tissuefactor independent activity of the polypeptide variant of the invention(a_(variant)) is also determined as the absorbance change after 10minutes. The ratio between the activity of the polypeptide variant, inits activated form, and the activity of rhFVIIa is defined asa_(variant)/a_(wt).

Clotting Assay

The clotting activity of the FVIIa and variants thereof were measured inone-stage assays and the clotting times were recorded on a ThrombotrackIV coagulometer (Medinor). Factor VII-depleted human plasma (AmericanDiagnostica) was reconstituted and equilibrated at room temperature for15-20 minutes. 50 microliters of plasma was then transferred to thecoagulometer cups.

FVIIa and variants thereof were diluted in Glyoxaline Buffer (5.7 mMbarbiturate, 4.3 mM sodium citrate, 117 mM NaCl, 1 mg/ml BSA, pH 7.35).The samples were added to the cup in 50 ul and incubated at 37° C. for 2minutes.

Thromboplastin (Medinor) was reconstituted with water and CaCl₂ wasadded to a final concentration of 4.5 mM. The reaction was initiated byadding 100 μl thromboplastin.

To measure the clotting activity in the absence of TF the same assay wasused without addition of thromboplastin. Data was analysed using PRISMsoftware.

Whole Blood Assay

The clotting activity of FVIIa and variants thereof were measured inone-stage assays and the clotting times were recorded on a ThrombotrackIV coagulometer (Medinor). 100 μl of FVIIa or variants thereof werediluted in a buffer containing 10 mM glycylglycine, 50 mM NaCl, 37.5 mMCaCl₂, pH 7.35 and transferred to the reaction cup. The clottingreaction was initiated by addition of 50 μL blood containing 10% 0.13 Mtri-sodium citrate as anticoagulant. Data was analysed using Excel orPRISM software.

Amidolytic Assay

The ability of the variants to cleave small peptide substrates can bemeasured using the chromogenic substrate S-2288(D-Ile-Pro-Arg-p-nitroanilide). FVIIa is diluted to about 10-90 nM inassay buffer (50 mM Na-Hepes pH 7.5, 150 mM NaCl, 5 mM CaCl₂, 0.1% BSA,1U/ml Heparin). Furthermore, soluble TF (sTF) is diluted to 50-450 nM inassay buffer. 120 μl of assay buffer is mixed with 20 μl of the FVIIasample and 20 μl sTF. After 5 min incubation at room temperature withgentle shaking, followed by 10 min incubation at 37° C., the reaction isstarted by addition of the S-2288 substrate to 1 mM and the absorptionat 405 nm is determined at several time points.

ELISA Assay

FVII/FVIIa (or variant) concentrations are determined by ELISA. Wells ofa microtiter plate are coated with an antibody directed against theprotease domain using a solution of 2 μg/ml in PBS (100 μl per well).After overnight coating at R.T. (room temperature), the wells are washed4 times with THT buffer (100 mM NaCl, 50 mM Tris-HCl pH 7.2 0.05%Tween-20). Subsequently, 200 μl of 1% Casein (diluted from 2.5% stockusing 100 mM NaCl, 50 mM Tris-HCl pH 7.2) is added per well forblocking. After 1 hr incubation at R.T., the wells are emptied, and 100μl of sample (optionally diluted in dilution buffer (THT+0.1% Casein))is added. After another incubation of 1 hr at room temperature, thewells are washed 4 times with THT buffer, and 100 μl of abiotin-labelled antibody directed against the EGF-like domain (1 μg/ml)is added. After another 1 hr incubation at R.T., followed by 4 morewashes with THT buffer, 100 μl of streptavidin-horse radish peroxidase(DAKO A/S, Glostrup, Denmark, 1/10000 diluted) is added. After another 1hr incubation at R.T., followed by 4 more washes with THT buffer, 100 μlof TMB (3,3′, 5,5′-tetramethylbenzidine, Kem-en-Tech A/S, Denmark) isadded. After 30 min incubation at R.T. in the dark, 100 μl of 1 M H₂SO₄is added and OD_(450 nm) is determined. A standard curve is preparedusing rhFVIIa (NovoSeven®).

Alternatively, FVII/FVIIa or variants may be quantified through the Gladomain rather than through the protease domain. In this ELISA set-up,wells are coated overnight with an antibody directed against theEGF-like domain and for detection, a calcium-dependent biotin-labelledmonoclonal anti-Gla domain antibody is used (2 μg/ml, 100 μl per well).In this set-up, 5 mM CaCl₂ is added to the THT and dilution buffers.

Thromobogram Assay

The effect of hFVIIa, rhFVIIa or FVIIa variants on thrombin generationin human plasma is tested in a modified version of the assay describedon page 589 in Hemker et al. (2000) Thromb Haemost 83:589-91. Briefly,the molecule to be assayed (either hFVIIa, rhFVIIa or a variant) ismixed with FVII-depleted platelet poor plasma (PPP) containing eitherrelipidated recombinant tissue factor (such as Innovin from DadeBehring) or phospholipid (phosphatidylcholine and phosphatidylserine ina ratio of 8:2, or phosphatidylcholine, phosphatidylserine andphosphatidylethanol in a ratio of 4:2:4).

The reaction is started by addition of a fluoregenic thrombin substrateand calcium chloride. The fluorescence is measured continuously and thethrombin amidolytic activity is determined by calculating the slope ofthe fluorescence curve (the increase in fluorescence over time). In thisway the time until maximum thrombin amidolytic activity is obtained(T_(max)), and the thrombin generation rate (maximal increase inthrombin activity) and the total thrombin work (area under the curve(AUC)) can be calculated.

Frozen citrated FVII-depeleted plasma is thawed in the presence of corntrypsin inhibitor (100 μg/ml serum) to inhibit the contact pathway ofcoagulation. To each well of a 96-well microtiter plate is added 80 μlplasma and 20 μl buffer containing rhFVII or variant to be tested infinal concentrations of between 0.1 and 100 nM. Recombinant human tissuefactor (rTF) is added in 5 μl assay buffer to a final concentration of 1pM. The assay buffer consists of 20 mM Hepes, 150 mM NaCl and 60 mg/mlBSA in distilled water. The reaction is started by adding 20 μl of thesubstrate solution containing 0.1 M calcium chloride. The assay plateand reagents are pre-warmed to 37° C. and the reaction takes place atthis temperature. The fluorimeter used is a BMG Fluormeter with anexcitation filter at 390 nm and an emission filter at 460 nm. Thefluorescence is measured in each well of 96-well clear bottom plates at20-40 second intervals over 30-180 minutes. Data are analyzed usingPRISM Software.

Tissue Factor Binding Surface Plasmon Resonance Assay (Biacore Assay)

Surface plasmon resonance analysis was used to determine the relativebinding of wild-type Factor VIIa and variants thereof to soluble tissuefactor. Recombinant soluble tissue factor that contains theextracellular domain was coupled to 270 response units on a Biacore CM5chip using NHS/EDC coupling. Soluble tissue factor was coupled at a pHof 4.5 to enable interaction with the chip surface.

In this assay, tissue factor binding of factor VII protein was comparedat a single concentration of FVIIa or variant to allow a relativecomparison of the variants to wild-type. This concentration wasdetermined by means of a standard curve of wild type FVIIa that wasflowed over the chip in concentrations between 75 and 0 μg/ml. FVIIa wasremoved by addition of 10 mM EDTA. It was determined in this manner thata concentration of 15 μg/ml gave binding in the linear range. Variantsof FVIIa were then flowed over the chip at 15 μg/ml to determine therelative binding strength of FVIIa or variants to tissue factor.

EXAMPLES Example 1

The X-ray structure of hFVIIa in complex with soluble tissue factor byBanner et al., J Mol Biol, 1996; 285:2089 is used for this example. Forfurther information on the calculations in this example, see WO01/58935.

Surface Exposure

Performing fractional ASA calculations resulted in the followingresidues being determined to have more than 25% of their side chainexposed to the surface: A1, N2, A3, F4, L5, E6, E7, L8, R9, P10, S12,L13, E14, E16, K18, E19, E20, Q21, S23, F24, E25, E26, R28, E29, F31,K32, D33, A34, E35, R36, K38, L39, W41, I42, S43, S45, G47, D48, Q49,A51, S52, S53, Q56, G58, S60, K62, D63, Q64, L65, Q66, S67, I69, F71,L73, P74, A75, E77, G78, R79, E82, T83, H84, K85, D86, D87, Q88, L89,I90, V92, N93, E94, G97, E99, S103, D104, H105, E99, S103, D104, H105,T106, G107, T108, K109, S111, R113, E116, G117, S119, L120, L121, A122,D123, G124, V125, S126, T128, P129, T130, V131, E132, I140, L141, E142,K143, R144, N145, A146, S147, K148, P149, Q150, G151, R152, G155, K157,V158, P160, K161, E163, L171, N173, G174, A175, N184, T185, I186, H193,K197, K199, N200, R202, N203, I205, S214, E215, H216, D217, G218, D219,S222, R224, S232, T233, V235, P236, G237, T238, T239, N240, H249, Q250,P251, V253, T255, D256, E265, R266, T267, E270, R271, F275, V276, R277,F278, L280, L287, L288, D289, R290, G291, A292, T293, L295, E296, N301,M306, T307, Q308, D309, L311, Q312, Q313, R315, K316, V317, G318, D319,S320, P321, N322, T324, E325, Y326, Y332, S333, D334, S336, K337, K341,G342, H351, R353, G354, Q366, G367, T370, V371, G372, R379, E385, Q388,K389, R392, S393, E394, P395, R396, P397, G398, V399, L400, L401, R402,P404 and P406 (A1-S45 are located in the Gla domain, the remainingpositions are located outside the Gla domain).

The following residues were determined to have more than 50% of theirside chain exposed to the surface: A1, A3, F4, L5, E6, E7, L8, R9, P10,E14, E16, K18, E19, E20, Q21, S23, E25, E26, E29, K32, A34, E35, R36,K38, L39, I42, S43, G47, D48, A51, S52, S53, Q56, G58, S60, K62, L65,Q66, S67, I69, F71, L73, P74, A75, E77, G78, R79, E82, H84, K85, D86,D87, Q88, L89, I90, V92, N93, E94, G97, T106, G107, T108, K109, S111,E116, S119, L121, A122, D123, G124, V131, E132, L141, E142, K143, R144,N145, A146, S147, K148, P149, Q150, G151, R152, G155, K157, P160, N173,G174, A175, K197, K199, N200, R202, S214, E215, H216, G218, R224, V235,P236, G237, T238, H249, Q250, V253, D256, T267, F275, R277, F278, L288,D289, R290, G291, A292, T293, L295, N301, M306, Q308, D309, L311, Q312,Q313, R315, K316, G318, D319, N322, E325, D334, K341, G354, G367, V371,E385, K389, R392, E394, R396, P397, G398, R402, P404 and P406 (A1-S43are located in the Gla domain, the remaining positions are locatedoutside the Gla domain).

Tissue Factor Binding Site

It was determined using ASA calculations that the following residues inhFVII change their ASA in the complex. These residues were defined asconstituting the receptor binding site: L13, K18, F31, E35, R36, L39,F40, I42, S43, S60, K62, D63, Q64, L65, I69, C70, F71, C72, L73, P74,F76, E77, G78, R79, E82, K85, Q88, I90, V92, N93, E94, R271, A274, F275,V276, R277, F278, R304, L305, M306, T307, Q308, D309, Q312, Q313, E325and R379.

Active Site Region

The active site region is defined as any residue having at least oneatom within a distance of 10 Å from any atom in the catalytic triad(residues H193, D242, S344): I153, Q167, V168, L169, L170, L171, Q176,L177, C178, G179, G180, T181, V188, V189, S190. A191, A192, H193, C194,F195, D196, K197, I198, W201, V228, I229, I230, P231, S232, T233, Y234,V235, P236, G237, T238, T239, N240, H241, D242, I243, A244, L245, L246,V281, S282, G283, W284, G285, Q286, T293, T324, E325, Y326, M327, F328,D338, S339, C340, K341, G342, D343, S344, G345, G346, P347, H348, L358,T359, G360, I361, V362, S363, W364, G365, C368, V376, Y377, T378, R379,V380, Q382, Y383, W386, L387, L400, and F405.

The Ridge of the Active Site Binding Cleft

The ridge of the active site binding cleft region was defined by visualinspection of the FVIIa structure 1FAK.pdb as: N173, A175, K199, N200,N203, D289, R290, G291, A292, P321 and T370.

Example 2

Design of an Expression Cassette for Expression of rhFVII in MammalianCells

The expression cassette for expression of rhFVII was designed and clonedas described in Example 2 of WO 01/58935.

Example 3

Construction of Expression Cassette Encoding Variants of the Invention

Sequence overhang extension (SOE) PCR was used for generating constructshaving variant FVII open reading frames with substituted codons by usingstandard methods.

Example 4

Expression of Polypeptide Variants in CHO K1 Cells

The cell line CHO K1 (ATCC # CCL-61) is seeded at 50% confluence in T-25flasks using MEMα, 10% FCS (Gibco/BRL Cat #10091), P/S and 5 μg/mlphylloquinone and allowed to grow until confluent. The confluent monocell layer is transfected with 5 μg of the relevant plasmid describedabove using the Lipofectamine 2000 transfection agent (LifeTechnologies) according to the manufacturer's instructions. Twenty fourhours post transfection a sample is drawn and quantified using e.g. anELISA recognizing the EGF1 domain of hFVII. At this time point relevantselection (e.g. Hygromycin B) may be applied to the cells with thepurpose of generating a pool of stable transfectants. When using CHO K1cells and the Hygromycin B resistance gene as selectable marker on theplasmid, this is usually achieved within one week.

Example 5

Generation of CHO K1 Cells Stably Expressing Polypeptide Variants.

A vial of CHO—K1 transfectant pool is thawed and the cells seeded in a175 cm² tissue flask containing 25 ml of MEMα, 10% FCS, phylloquinone (5μg/ml), 100 U/l penicillin, 100 μg/l streptomycin and grown for 24hours. The cells are harvested, diluted and plated in 96-well microtiterplates at a cell density of ½-1 cell/well. After a week of growth,colonies of 20-100 cells are present in the wells and those wellscontaining only one colony are labelled. After a further two weeks, themedia in all wells containing only one colony is substituted with 200 μlfresh medium. After 24 hours, a medium sample is withdrawn and analysedby e.g. ELISA. High producing clones are selected and used for largescale production of FVII or variants.

Example 6

Purification of Polypeptide Variants and Subsequent Activation

FVII and FVII variants are purified as follows: The procedure isperformed at 4° C. The harvested culture media from large-scaleproduction is ultrafiltered and subsequently diafiltered against 10 mMTris pH 8.6, using a Millipore TFF system with 30 kDa cut-off Pelliconmembranes. After concentration of the medium, citrate is added to 5 mMand the pH is adjusted to 8.6. If necessary, the conductivity is loweredto below 10 mS/cm. Alternatively, the media may be diluted and pH- andcitrate-adjusted without prior ultra- and diafiltration. Subsequently,the sample is applied to a Q-sepharose FF column, equilibrated with 50mM NaCl, 10 mM Tris pH 8.6. After washing the column with 100 mM NaCl,10 mM Tris pH 8.6, and for some variants 150 mM NaCl, 10 mM Tris pH 8.6,FVII is eluted using 10 mM Tris, 25 mM NaCl, 35 mM CaCl₂, pH 8.6. In theelution step, the concentration of CaCl₂ or NaCl can be increased ifnecessary to improve the yield.

For the second chromatographic step, an affinity column is prepared bycoupling of a monoclonal Calcium-dependent antiGla-domain antibody toCNBr-activated Sepharose FF. About 5.5 mg antibody is coupled per mlresin. The column is equilibrated with 10 mM Tris, up to 100 mM NaCl, 35mM CaCl₂, pH 7.5. NaCl is for some variants added to the sample to aconcentration of 100 mM NaCl and the pH is adjusted to 7.4-7.6. AfterO/N application of the sample, the column is washed with up to 100 mMNaCl, 35 mM CaCl₂, 10 mM Tris pH 7.5, and the FVII protein is elutedwith 100 mM NaCl, 50 mM citrate, 75 mM Tris pH 7.5, or alternatively 10mM Tris, 25 mM NaCl, 5 mM EDTA pH 8.6. The latter elution buffer allowsfor a direct load onto the third chromatographic column without anyadjustment of the eluate.

For the third chromatographic step, the conductivity of the sample islowered to below 10 mS/cm, if necessary, and the pH is adjusted to 8.6.The sample is then applied to an anion exchange column, typically aQ-sepharose column at a density around 3-10 mg protein per ml gel or aPoros 50 HQ column (Applied BioSciences) at a density of 5-40 mg proteinper ml gel to obtain efficient activation, both columns previouslyequilibrated with 25-50 mM NaCl, 10 mM Tris pH 8.6. After application,the column is washed with up to 50 mM NaCl, 0.25 mM CaCl₂, 10 mM Tris pH8.6 for 2-4 hours with a flow of 3-4 column volumes (cv) per hour. TheFVII protein is eluted using a gradient of 0-100% of 500 mM NaCl, 10 mMTris pH 8.6 over 40 cv. FVII containing fractions are pooled.

For the final purification step either an anion exchange or agelfiltration step isused. For the anion exchange chromatographic step,the conductivity is lowered to below 10 mS/cm. Subsequently, the sampleis applied to a Q-sepharose column (equilibrated with 140 mM NaCl, 10 mMglycylglycine pH 8.6) at a concentration of 3-5 mg protein per ml gel.The column is then washed with 140 mM NaCl, 10 mM glycylglycine pH 8.6and FVII is eluted with 140 mM NaCl, 15-35 mM CaCl₂, 10 mM glycylglycinepH 8.6. The eluate is diluted to 10 mM CaCl₂ and the pH is adjusted6.8-7.2. Finally, Tween-80 is added to 0.01% and the pH is adjusted to5.5 for storage at −80° C. For the gel filtration step, a G25 column(HiPrep, Amersham Biosciences) is equilibrated and run in 10 mMglycylglycine, 10 mM CaCl₂, 140 mM NaCl, 0.01% Tween-80, pH 5.5.

Example 7

Experimental Results—FX Activation Activity

Subjecting the variants of the invention to the “TF-independent Factor XActivation Assay”, the following results were obtained (the resultsbeing expressed as a percentage of the activity of the P10Q+K32E variantas a reference): TABLE 1 TF-independent FX activation Variant(a_(variant)/a_(P10QK32E)) * 100 rhFVIIa 10 P10Q + K32E (reference) 100A3AY + P10Q + K32E + A34L 216 P10Q + K32E + D33F + A34E 194 P10Q +K32E + A34E + P74S 190 P10Q + K32E + A34E + R36E + K38E 144 P10Q +K32E + A34D + R36E 140 P10Q + K32E + A34E + R36E + 51 T106N + V253N

As it appears from the above results, the variants of the inventionshowed a substantial improvement in FX activation activity as comparedto rhFVIIa and also as compared to [P10Q+K32E]rhFVIIa.

Example 8

Experimental Results—Clotting Activity in the “Whole Blood Assay”

Subjecting variants of the invention to the “Whole Blood Assay” revealedthat they exhibited a significantly increased clotting activity (i.e.reduced clotting time) as compared to rhFVIIa as well as[P10Q+K32E]rhFVIIa. The experimental results are shown in FIG. 1 andTable 2 below. TABLE 2 Clotting time (Whole Blood Assay) Variantt_(variant)/t_(wt) rhFVIIa (reference) 1 A3AY + P10Q + K32E + E116D 0.4A3AY + P10Q + K32E + A34L 0.3 P10Q + K32E + A34E + P74S 0.3 A3AY +P10Q + K32E + E77A 0.4 P10Q + K32E + A34E + R36E + 0.2 T106N + V253N

Example 9

Experimental Results—Clotting Activity in the “Clotting Assay”

When assayed in a TF-dependent clotting assay (the “Clotting Assay”described above in the Materials and Methods section) it was evidentthat variants of the invention having the R36E substitution have asignificantly reduced clotting activity when compared to rhFVII or toother variants of the invention. See Table 3 below. Nevertheless, asillustrated in Example 7 above, variants having the R36E substitutionhave an increased Factor X activation activity in the “TF-independentFactor X Activation Assay”. TABLE 3 Average Clotting Activity(units/mg_(variant)/units/mg_(wt)) Variant (n = 2-3) NovoSeven ®(reference) 52,119 (100%) P10Q + K32E 52,714 (101%) A3AY + P10Q + K32E +A34L 56,948 (107%) P10Q + K32E + A34E + R36E  1,439 (2.7%) P10Q + K32E +A34D + R36E + K38E  1,232 (2.4%) P10Q + K32E + A34E + R36E +   180 (<1%)T106N + V253N

Example 10

Experimental Results—Throbim Generation in the Thrombogram Assay

Using both phospholipid(PL)-dependent and tissue factor(TF)-dependentthrombograms (see the description of the Thrombogram Assay above), themaximum rate of thrombin generation was determined for FVIIa variants atdifferent concentrations of variant proteins. By plotting the maximumthrombin generation rates (expressed as FU (fluorescence units) persec²) as a function of the variant concentration in pM, the resultsshown in FIG. 2 (maximum tissue factor-dependent thrombin generationrate) and FIG. 3 (maximum phospholipid-dependent thrombin generationrate) were obtained.

From these results it is evident that the FVIIa variant P10Q K32E A34ER36E has a differentiated thrombin generation ability depending on thewhether the reaction is PL-dependent or TF-dependent. The maximumTF-dependent thrombin generation rate of this variant is decreased byapproximately 10-fold (punctuated line in FIG. 2) when compared to theFVIIa variants P10Q K32E or A3AY P10Q K32E A34L. Also, lag time, time topeak, peak height and (to a lesser extent) AUC are reduced for P10Q K32EA34E R36E compared to the other variants (results not shown). Incontrast to the TF-dependent activity, the PL-dependent activity of theP10Q K32E A34E R36E variant is equivalent to that of the other variantstested in this example (see FIG. 3), i.e. this variant has fullPL-dependent activity even though the TF-dependent activity issubstantially reduced.

In the same experiment, the variant P10Q K32E A34E R36E was compareddirectly to the variant P10Q K32E A34E P74S, which has a highTF-dependent thrombin generation rate as shown in FIG. 2. Thedifferences in TF-binding between these two variants (i.e. the reducedTF-binding of the variant P10Q K32E A34E R36E) is believed to bedirectly attributable to the presence of the R36E substitution, possiblyin synergy with the A34E substitution.

In addition, the PL-dependent and TF-dependent activity of the variantP10Q+K32E+A34E+R36E+T106N+V253N was compared to that of wild-typerhFVIIa (NovoSeven®). The results, in the form of thrombogramsillustrating thrombin generation as a function of time, are shown inFIGS. 4 (PL-dependent activity) and 5 (TF-dependent activity).

FIG. 4 shows that the PL-dependent activity of the variant of theinvention, compared to rhFVIIa, has a reduced lag time, a reduced timeto peak, an increased peak height, and an increased maximum rate ofthrombin generation. In contrast to the results for the PF-dependentactivity, FIG. 5 shows that the TF-dependent activity of the variant ofthe invention, compared to rhFVIIa, has an increased lag time, anincreased time to peak, a reduced peak height, and a reduced maximumrate of thrombin generation.

Taken together, the results illustrated in FIGS. 4 and 5 show that thevariant of the invention, P10Q+K32E+A34E+R36E+T106N+V253N, has anenhanced phospholipid-dependent activity and at the same time a reducedtissue factor-dependent activity compared to rhFVIIa. Both of theseproperties are contemplated to be advantageous in a clinical setting,e.g. in the case of trauma or intracerebral haemorrhage, the enhancedPL-dependent activity for obtaining faster and more effective bloodclotting, and the reduced TF-dependent activity for minimizing the riskof undesired blood clot formation.

Example 11

Experimental Results—FVIIa Binding to Tissue Factor in the Biacore Assay

Subjecting variants of the invention to assay by surface plasmonresonance on a Biacore system using a TF chip as described in theMaterials and Methods section, the following results were obtained:TABLE 4 Average response units Variant (n = 5) Wild-type FVIIa 888 P10Q;K32E 714 A3AY; P10Q; K32E; A34L  967* P10Q; K32E; A34E; R36E 414*n = 2

In consistency with the TF-dependent thrombin generation rate data fromthe Thrombogram Assay (Example 10), the results in Table 4 indicate thatthe R36E substitution confers less binding to tissue factor.

In the same Biacore Assay, FVIIa variants having the same modificationsas the variants listed in Table 4 together with two additionalmodifications introducing two glycosylation sites (T106N and eitherV253N or I205T) were also tested for binding to tissue factor. Theresults are shown in Table 5 below. TABLE 5 Average response unitsVariant (n = 5) T106N; V253N 717 T106N; I205T 612 P10Q; K32E; T106N;I205T 502 P10Q; K32E; T106N; V253N 498 A3AY; P10Q; K32E; A34L; T106N;V253N 522 P10Q; K32E; A34E; R36E; T106N; I205T 216

These results are consistent with those of Table 4 and show thatcompared to the same variants (or the wild-type) in Table 4 without theadditional glycosylation sites, the presence of two new glycosylationsites in the variants of Table 5 provides a (further) reduction intissue factor binding. As was the case for the variants of Table 4, thepresence of the R36E substitution in a glycosylation variant alsoresults in a level of tissue factor binding that is substantially lowerthan the tissue factor binding of the other glycosylation variants thatdo not have this substitution.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. It is understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and scope of the appended claims. For example, all thetechniques and apparatus described above may be used in variouscombinations. All publications, patents, patent applications, and/orother documents cited in this application are incorporated herein byreference in their entirety for all purposes to the same extent as ifeach individual publication, patent, patent application, and/or otherdocument were individually indicated to be incorporated herein byreference in its entirety for all purposes.

1.-25. (canceled)
 26. A method for treating a mammal having a disease ora disorder wherein clot formation is desirable, comprising administeringto a mammal in need thereof an effective amount of a Factor VII (FVII)or Factor VIIa (FVIIa) polypeptide variant having an amino acid sequencecomprising 1-15 amino acid modifications relative to human Factor VII(hFVII) or human Factor VIIa (hFVIIa) with the amino acid sequence shownin SEQ ID NO:1. the variant comprising an amino acid substitution inposition
 36. 27. The method of claim 26, wherein said disease ordisorder is selected from the group consisting of hemorrhages, includingbrain hemorrhages, severe uncontrolled bleedings, such as trauma,bleedings in patients undergoing transplantations or resection, varicealbleeding, and hemophilia.
 28. The method of claim 27, wherein saiddisease or disorder is trauma.
 29. The method of claim 27, wherein saiddisease or disorder is hemophilia.
 30. A method for treatingintracerebral haemorrhage or traumatic brain injury, comprisingadministering to a patient in need thereof an effective amount of aFactor VII (FVII) or Factor VIIa (FVIIa) polypeptide variant having anamino acid sequence comprising 1-15 amino acid modifications relative tohuman Factor VII (hFVII) or human Factor VIIa (hFVIIa) with the aminoacid sequence shown in SEQ ID NO:1, the variant comprising an amino acidsubstitution in position 36.